Non-human mammals for the production of chimeric antibodies

ABSTRACT

The invention provides knock-in non-human cells and mammals having a genome encoding chimeric antibodies and methods of producing knock-in cells and mammals. Certain aspects of the invention include chimeric antibodies, humanized antibodies, pharmaceutical compositions and kits. Certain aspects of the invention also relate to diagnostic and treatment methods using the antibodies of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/121,883, filed Jun. 9, 2011, which is a U.S. National stageapplication filed under 35 U.S.C. §371 of International PatentApplication No. PCT/US2009/059131, accorded an international filing dateof Sep. 30, 2009, which claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application No. 61/101,938 filed Oct. 1, 2008and U.S. Provisional Patent Application No. 61/101,597 filed Sep. 30,2008, where these applications are incorporated herein by reference intheir entireties.

BACKGROUND

1. Technical Field

The present invention is directed generally to the production ofknock-in non-human mammals and cells and chimeric immunoglobulin chainsand antibodies.

2. Description of the Related Art

Disease therapies utilizing monoclonal antibodies (mAbs) haverevolutionized medicine, and mAb-based drugs are now utilized in thetreatment of cancer, autoimmunity, inflammation, macular degeneration,infections, etc. However, the available technologies for generation anddiscovery of mAbs for use in the prevention and treatment of diseasesand disorders have significant drawbacks including inefficiency, absenceor loss of sufficient potency, absence or loss of specificity and theinduction of an immune response against the therapeutic mAb. The firstattempts to use mAbs as therapeutics were hindered by the immunogenicityof the mouse amino acid composition of the mAbs. When administered tohumans, the mouse amino acid sequence elicited a human anti-mouseantibody (HAMA) response that dramatically reduced the potency andpharmacokinetics of the drug as well as causing severe and potentiallyfatal allergic reactions.

Additional methods to generate mAb therapeutics include chimerized mAbs(cmAbs) created through recombinant DNA technology combining amouse-derived variable domain appended to a human constant region. Othermethods of generating antibodies involve humanizing mAbs in vitro tofurther reduce the amount of mouse amino acid sequence in a therapeuticmAb. Antibody-display technologies developed to generate “fully-human”antibodies in vitro have yet to adequately mimic the natural antibodymaturation process that occurs during an in vivo immune response (seepg. 1122-23, Lonberg, Nat. Biotech. (2005) 23:1117-1125.) mAbs developedusing these methods can elicit an immune response that can reduceefficacy and/or be life-threatening, and they are typically atime-consuming and costly process. Also, during the molecular processesinherent in these methods, loss of affinity and epitope shifting canoccur, thereby reducing potency and introducing undesirable changes inspecificity.

Transgenic mice have been engineered to produce fully human antibodiesby introducing human antibody transgenes to functionally replaceinactivated mouse immunoglobulin (Ig) loci. However, many of thesetransgenic mouse models lack important components in the antibodydevelopment process, such as sufficient diversity in the genes fromwhich antibody variable regions are generated, the ability to make IgD(Loset et al., J. Immunol., (2004) 172:2925-2934), important cisregulatory elements important for class switch recombination (CSR), or afully functional 3′ locus control region (LCR) (e.g., U.S. Pat. No.7,049,426; and Pan et al., Eur. J. Immunol. (2000) 30:1019-1029). Sometransgenic mice contain yeast artificial chromosomes or human minilocias integrated transgenes. Others carry transchromosomes that exhibitvarious frequencies of mitotic and meiotic instability. Furthermore, thefully human constant regions of these transgenic mice functionsub-optimally due to reduced activity in conjunction with otherendogenous and trans-acting components of the BCR signal transductionapparatus, e.g., Igα and Igβ, and Fc receptors (FcR), as compared tonormal mice.

Mice have also been genetically engineered to produce chimericantibodies that are composed of human V domains appended to mouse Cdomains that remain fully intact, with the fully-intact and modifiedportions including all genomic DNA downstream of the J gene cluster (seeU.S. Pat. Nos. 5,770,429 and 6,596,541 and U.S. Patent ApplicationPublication No. 2007/0061900). Human V regions from these mice can berecovered and appended to human constant region genes by molecularbiological methods and expressed by recombinant methods to producefully-human antibodies. The antibodies from these mice may exhibitreduction or loss of activity, potency, solubility etc. when the human Vregion is removed from the context of the mouse C domains with which itwas evolved and then appended to a human C region to make a fully humanantibody.

Current methods of developing a therapeutic mAb can alter functions ofthe antibody, such as solubility, potency and antigen specificity, whichwere selected for during initial stages development. In addition, mAbsgenerated by current methods have the potential to elicit a dangerousimmune response upon administration. Current human and chimeric antibodyproducing mice lack appropriate genetic content to function properly,e.g., genetic diversity, cis regulatory elements, trans actingregulatory elements, signaling domains, genetic stability. It would bebeneficial to develop methods and compositions for the enhancedgeneration and discovery of therapeutic antibodies and that retainpotency and specificity through the antibody generation, discovery,development, and production process without eliciting an immuneresponse, as well as methods of producing such antibodies. The presentinvention provides a solution for generating such antibodies intransgenic animals.

BRIEF SUMMARY

The present invention relates to knock-in non-human mammals and cells,antibodies, methods, compositions (including pharmaceuticalcompositions) as well as kits of various embodiments disclosed herein.More specifically, the present invention relates to methods,compositions and kits relating to chimeric Ig chains and antibodiesproduced by the knock-in non-human mammals and cells and the humanantibodies and fragments thereof engineered from the variable domains ofsaid chimeric antibodies.

Some aspects of the invention relate to a homologous recombinationcompetent non-human mammalian cell having a genome comprising (1) ahuman VH gene segment and (2) a portion of a syngeneic Ig heavy chainlocus comprising all or a part of gene segments downstream of JH whereina syngeneic CH1 domain is replaced with a human CH1 domain and whereinthe human VH gene segment and the syngeneic Ig heavy chain locus replacean endogenous Ig heavy chain, or a portion thereof, so that the cellcomprises a genome encoding a chimeric Ig heavy chain. In certainembodiments, the portion further comprises a 3′ locus control region(LCR), or a functional fragment thereof. In one embodiment, the cellfurther comprises a human DH gene segment. In another embodiment, thecell further comprises a DH gene segment from a mammal. In a particularembodiment, the DH gene segment is selected from the group consisting ofhuman, non-human primate, rabbit, sheep, rat, hamster, and mouse. Inparticular embodiments, the DH gene segment is human. In anotherembodiment, the human CH1 domain comprises a single CH gene segment,e.g., Cγ1, Cγ2, and Cγ4.

In yet another embodiment, the cell further comprises a human upperhinge gene segment. In a related embodiment, the cell further comprisesa human middle hinge gene segment. In particular embodiments, the humanhinge gene segment is an IgG4 hinge gene segment. In some embodiments,serine is substituted for the proline at position 229. In certainpreferred embodiments, the chimeric Ig heavy chain is capable of bindingan endogenous FcR.

In certain embodiments, the cell is an embryonic stem cell. In anotherembodiment, the cell is a mouse cell.

Another aspect of the invention relates to a method of producing thecell described above that comprises a genome encoding a chimeric Igheavy chain comprising the steps of producing a first BAC comprising ahuman VH gene segment; producing a second BAC comprising a portion of asyngeneic Ig heavy chain locus comprising all or a part of the genesegments downstream of JH, wherein a syngeneic CH1 domain is replacedwith a human CH1 domain; introducing the first BAC into a homologousrecombination competent non-human mammalian cell and replacing all or aportion of an endogenous VH gene segment via homologous recombination;and introducing the second BAC into the cell and replacing all or aportion of an endogenous Ig heavy chain locus via homologousrecombination, so that the cell comprises a genome encoding a chimericIg heavy chain. In one embodiment, either the first or second BACfurther comprises a human JH gene segment.

In particular embodiments, the first BAC further comprises a firstsite-specific recombinase recognition sequence near the 3′ end of thefirst BAC, and the second BAC further comprises a second site-specificrecombinase recognition sequence near the 5′ end of the second BAC. Arelated embodiment further comprises the step of expressing asite-specific recombinase, wherein an intervening sequence between thefirst and second site-specific recombinase recognition sequences isremoved. In a particular embodiment, the first and second site-specificrecombinase recognition sequences are loxP or variants thereof, and thesite-specific recombinase is CRE. In another embodiment, the first andsecond site-specific recombinase recognition sequences are frt, and thesite-specific recombinase is flp.

In certain embodiments related to a method for producing the cellaccording to the invention, the introducing step of the second BACoccurs before the introducing step of the first BAC.

Some aspects of the invention relate to a homologous recombinationcompetent non-human mammalian cell having a genome comprising a human Iglight chain locus, or a portion thereof, wherein the human Ig lightchain locus replaces all or a portion of an endogenous Ig light chainlocus, so that the cell comprises a genome encoding a human Ig lightchain, or a portion thereof. In one embodiment, the human Ig light chainlocus comprises a human Igκ variable region. In a related embodiment,the Ig light chain locus further comprises a human Igκ constant region.

In yet another embodiment, the human Ig light chain locus comprises allor a portion of a human Igλ light chain locus and an Igλ 3′LCR, or afunctional fragment thereof. In one embodiment, the human Igλ lightchain locus comprises the entire human Igλ locus. In another embodimentthe human Igλ light chain locus comprises human Vλ gene segments and 1to 7 Jλ-Cλ gene segment pairs, wherein the human Cλ is replaced withsyngeneic Cλ. In yet another embodiment, the human Igλ light chain locuscomprises human Vλ gene segments, 1 to 7 human Jλ gene segments, and asingle human Cλ gene segment, wherein the human gene segments resemble ahuman Igλ locus configuration. In particular embodiments, the Igλ 3′LCR, or a functional fragment thereof, is from a mammal selected fromthe group consisting of human, non-human primate, and rat. In oneembodiment the Igλ 3′ LCR, or a functional fragment thereof, is human.In particular embodiments, the Igλ 3′ LCR, or a functional fragmentthereof, binds NFκb. In one embodiment, the Igλ 3′ LCR, or a functionalfragment thereof, is from mouse and has been mutagenized so as torestore binding of NFκb. In other embodiments, the 3′ LCR, or afunctional fragment thereof, in the human Igλ locus is an Igκ 3′ LCR, orfunctional fragment thereof.

In certain embodiments, the cell is an embryonic stem cell. In anotherembodiment, the cell is a mouse cell.

Another aspect of the invention relates to a method of producing thecell described above that comprises a genome encoding a human Ig lightchain, or a portion thereof, comprising the steps of producing a firstBAC comprising a human Ig light chain locus, or a portion thereof;introducing the first BAC into a homologous recombination competentnon-human mammalian cell; and replacing an endogenous Ig light chainlocus, or a portion thereof, via homologous recombination, so that thecell comprises a genome encoding a human Ig light chain, or a portionthereof.

In particular embodiments, the human Ig light chain locus comprises ahuman Igκ variable region. A related embodiment further comprises thesteps of producing a second BAC comprising a human Igκ constant region;introducing the second BAC into the cell; and replacing all or a portionof an endogenous Igκ constant region.

In yet another embodiment, the first BAC further comprises a firstsite-specific recombinase recognition sequence near the 3′ end of thefirst BAC, and the second BAC further comprises a second site-specificrecombinase recognition sequence near the 5′ end of the second BAC. In arelated embodiment, the method further comprises the step of expressinga site-specific recombinase, wherein an intervening sequence between thefirst and second site-specific recombinase recognition sequences isremoved. In a particular embodiment, the first and second site-specificrecombinase recognition sequences are loxP or variants thereof, and thesite-specific recombinase is CRE. In another embodiment, the first andsecond site-specific recombinase recognition sequences are frt, and thesite-specific recombinase is flp.

In certain embodiments related to a method for producing the cellaccording to the invention, the introducing step of the second BACoccurs before the introducing step of the first BAC.

In certain embodiments, the human Ig light chain locus comprises all ora portion of a human Igλ light chain locus and an Igλ 3′LCR, or afunctional fragment thereof. In one embodiment, the human Igλ lightchain locus comprises a human Igλ light chain variable region. A relatedembodiment further comprises the steps of producing a second BACcomprising a human Igλ constant region; introducing the second BAC intothe cell; and replacing all or a portion of an endogenous Igλ constantregion. In some embodiments, the first BAC further comprises a firstsite-specific recombinase recognition sequence near the 3′ end of thefirst BAC, and the second BAC further comprises a second site-specificrecombinase recognition sequence near the 5′ end of the second BAC. In arelated embodiment, the method further comprises the step of expressinga site-specific recombinase, wherein an intervening sequence between thefirst and second site-specific recombinase recognition sequences isremoved. In a particular embodiment, the first and second site-specificrecombinase recognition sequences are loxP or variants thereof, and thesite-specific recombinase is CRE. In another embodiment, the first andsecond site-specific recombinase recognition sequences are frt, and thesite-specific recombinase is flp.

In certain embodiments, the human Igλ light chain locus comprises humanVλ gene segments and 1 to 7 Jλ-Cλ gene segment pairs, wherein the humanCλ is replaced with syngeneic Cλ. In yet another embodiment, the humanIgλ light chain locus comprises human Vλ gene segments, 1 to 7 human Jλgene segments, and a single human Cλ gene segment, wherein said humangene segments resemble a human Igλ locus configuration. A related methodfurther comprises confirming the incorporation of splice sequences intothe BAC, and incorporating the splice sequences if not alreadyincorporated into the BAC, prior to the introducing step.

In one embodiment, the human Vλ gene segments comprise cluster A. Arelated embodiment further comprises the steps of producing a second BACcomprising cluster B human Vλ gene segments; introducing the second BACinto said cell; and replacing endogenous Vλ gene segments. In a furtherembodiment, the second BAC further comprises human cluster C Vλ genesegments.

Some aspects of the invention relate to a homologous recombinationcompetent non-human mammalian cell having a genome comprising (1) ahuman Ig locus, or a portion thereof, and (2) a cluster of human FcRgenes, wherein the human Ig locus and FcR genes replace the endogenousregions, and wherein the genome encodes a human Ig chain, or a portionthereof, and a human FcR. In particular embodiments, the human Ig locuscomprises a portion of a human Ig heavy chain locus comprising all or apart of the gene segments downstream of JH, such that the portion of ahuman Ig heavy chain engages a human FcR. In certain embodiments, thecell is a non-human embryonic stem cell. In other embodiments, the cellis a mouse cell.

Certain aspects of the invention relate to a method of producing thecell described above that has a genome encodes a human Ig chain, or aportion thereof, and a human FcR comprising the steps of producing afirst BAC comprising a human Ig locus, or a portion thereof; producing asecond BAC comprising a cluster of human FcR genes; introducing thefirst BAC into a homologous recombination competent non-human mammaliancell and replacing all or a portion of an endogenous Ig locus viahomologous recombination; and introducing the second BAC into said celland replacing an endogenous cluster of FcR genes via homologousrecombination, so that the cell comprises a genome encoding a human Igchain, or a portion thereof, and a human FcR.

Another aspect of the invention relates to a knock-in non-human mammalhaving a genome comprising (1) a human VH gene segment and (2) a portionof a syngeneic Ig heavy chain locus comprising all or a part of genesegments downstream of JH wherein a syngeneic CH1 domain is replacedwith a human CH1 domain; and wherein the human VH gene segment and thesyngeneic Ig heavy chain locus replace all or a portion of an endogenousIg heavy chain locus, so that the mammal is capable of producing achimeric Ig heavy chain.

In one embodiment, the mammal further comprises a human JH gene segment.In certain embodiments, the portion further comprises a 3′ locus controlregion (LCR), or a functional fragment thereof. In another embodiment,the mammal further comprises a DH gene segment from a mammal. In aparticular embodiment, the DH gene segment is selected from the groupconsisting of human, non-human primate, rabbit, sheep, rat, hamster, andmouse. In particular embodiments, the DH gene segment is human. Inanother embodiment, the human CH1 domain comprises a single CH genesegment, e.g., Cγ1, Cγ2, and Cγ4.

In yet another embodiment, the mammal further comprises a human upperhinge gene segment. In a related embodiment, the cell further comprisesa human middle hinge gene segment. In particular embodiments, the humanhinge gene segment is an IgG4 hinge gene segment. In some embodiments,serine is substituted for the proline at position 229. In certainpreferred embodiments, the chimeric Ig heavy chain is capable of bindingan endogenous FcR.

Yet another aspect of the invention relates to a knock-in non-humanmammal having a genome comprising a human Ig light chain locus, or aportion thereof, wherein said human Ig light chain locus replaces all ora portion of an endogenous Ig light chain locus, such that said mammalis capable of producing a human Ig light chain, or a portion thereof.

In one embodiment, the human Ig light chain locus comprises a human Igκvariable region. In a related embodiment, the Ig light chain locusfurther comprises a human Igκ constant region.

In yet another embodiment, the human Ig light chain locus comprises allor a portion of a human Igλ light chain locus and an Igλ 3′LCR, or afunctional fragment thereof. In one embodiment, the human Igλ lightchain locus comprises the entire human Igλ locus. In another embodimentthe human Igλ light chain locus comprises human Vλ gene segments and 1to 7 Jλ-Cλ gene segment pairs, wherein the human Cλ is replaced withsyngeneic Cλ. In yet another embodiment, the human Igλ light chain locuscomprises human Vλ gene segments, 1 to 7 human Jλ gene segments, and asingle human Cλ gene segment, wherein the human gene segments resemble ahuman Igλ locus configuration. In a related embodiment, the chimericantibody rearranges and expresses, resulting in a representation of Igλrelative to Igκ greater than 40:60. In particular embodiments, the Igλ3′ LCR, or a functional fragment thereof, is from a mammal selected fromthe group consisting of human, non-human primate, and rat. In oneembodiment the Igλ 3′ LCR, or a functional fragment thereof, is human.In particular embodiments, the Igλ 3′ LCR, or a functional fragmentthereof, binds NFκb. In one embodiment, the Igλ 3′ LCR, or a functionalfragment thereof, is from mouse and has been mutagenized so as torestore binding of NFκb. In other embodiments, the 3′ LCR, or afunctional fragment thereof, in the human Igλ locus is an Igκ 3′ LCR, orfunctional fragment thereof.

In certain embodiments, the knock-in non-human mammal capable ofproducing a chimeric Ig heavy chain described above further comprises ahuman Ig light chain locus, or a portion thereof, wherein the human Iglight chain locus replaces all or a portion of an endogenous Ig lightchain locus.

Another aspect of the invention provides a knock-in non-human mammalhaving a genome comprising (1) a human Ig locus, or a portion thereof,and (2) a cluster of human FcR genes, wherein the human Ig locus and FcRgenes replace the orthologous endogenous regions, and wherein the mammalis capable of producing a human Ig chain, or a portion thereof, and ahuman FcR. In one embodiment, the human Ig locus comprises a portion ofa human Ig heavy chain locus, said portion comprising all or a part ofthe gene segments downstream of JH, such that said Ig heavy chainengages a human FcR. In a related embodiment, the human Ig heavy chainlocus further comprises all or a part of a human variable region. In yetanother embodiment, the mammal further comprises a human Ig light chainlocus, or a portion thereof, wherein said human Ig light chain locusreplaces an endogenous Ig light chain locus, or a portion thereof, suchthat the knock-in non-human mammal is capable of producing a humanantibody and a human FcR.

In particular embodiments, the knock-in non-human mammal according tothe invention is a mouse.

Another embodiment relates to an antibody produced by the knock-innon-human mammal according to the invention, wherein said antibodycomprises a human Ig heavy or light chain, or a portion thereof. Oneembodiment provides a method of producing an antibody that specificallybinds to a target antigen comprising immunizing a knock-in non-humanmammal according to the invention with the target antigen and recoveringthe antibody that comprises a human Ig heavy or light chain, or aportion thereof.

Yet another embodiment provides a method of detecting a target antigencomprising detecting an antibody according to the invention with asecondary detection agent that recognizes a portion of the antibody. Inrelated embodiments, the portion comprises the Fc region of theantibody, a CH1 domain, a CH2 domain, a CH3 domain, the Fab domain ofthe antibody, the F(ab′)₂ domain of the antibody, a Cκ domain, or a Cλdomain of the antibody. In another embodiment, the method furthercomprises evaluating tissue distribution of said target antigen.

Certain embodiments relate to a pharmaceutical composition comprisingthe antibody according to the invention and a pharmaceuticallyacceptable carrier. In another embodiment, a kit comprises the antibodyaccording to the invention and instructions for use of said antibody.

Another aspect of the invention relates to a humanized antibody encodedby a polynucleotide sequence comprising (1) a polynucleotide sequenceencoding the human gene segments of the antibody according to theinvention appended to (2) a polynucleotide sequence encoding theremaining portion of a human constant region. In one embodiment, thehuman gene segments comprise human V region gene segments. In anotherembodiment, a pharmaceutical composition comprises the humanizedantibody and a pharmaceutically acceptable carrier. In yet anotherembodiment, a kit comprises the humanized antibody and instructions foruse of said antibody.

Yet another aspect of the invention relates to a method of producing themammal capable of producing a chimeric Ig heavy chain described abovecomprising the steps of producing a first BAC comprising a human VH genesegment; producing a second BAC comprising a portion of a syngeneic Igheavy chain locus comprising all or a part of gene segments downstreamof JH, wherein a syngeneic CH1 domain is replaced with a human CH1domain; introducing the first BAC into a homologous recombinationcompetent non-human mammalian cell and replacing an endogenous VH genesegment via homologous recombination; introducing the second BAC intosaid cell and replacing all or a portion of an endogenous Ig heavy chainlocus via homologous recombination; and generating from the cell aknock-in non-human mammal capable of producing a chimeric Ig heavychain. In one embodiment, either the first or second BAC furthercomprises a human JH gene segment. In certain embodiments, thegenerating step comprises blastocyst microinjection, morula aggregation,or somatic cell nuclear transfer.

Another aspect of the invention relates to a method of producing themammal capable of producing a human Ig light chain described abovecomprising the steps of producing a first BAC comprising a human Iglight chain locus, or a portion thereof; introducing the first BAC intoa homologous recombination competent non-human mammalian cell andreplacing all or a portion of an orthologous endogenous Ig light chainlocus via homologous recombination; and generating from the cell aknock-in non-human mammal capable of producing a human Ig light chain,or a portion thereof.

In certain embodiments, the human Ig light chain locus comprises an Igκvariable region. A further embodiment comprises the steps of producing asecond BAC comprising a human Igκ constant region; introducing thesecond BAC into the cell; and replacing all or a portion of anendogenous Igκ constant region.

In certain embodiments related to a method for producing the mammalaccording to the invention, the introducing step of the second BACoccurs before the introducing step of the first BAC.

In one embodiment, the human Ig light chain locus comprises all or aportion of a human Igλ light chain locus and an Igλ 3′LCR, or afunctional fragment thereof, replacing an orthologous endogenous Igλlocus and 3′ LCR via homologous recombination. In a related embodiment,the Igλ is expressed at about a ratio of 40:60 relative to Igκ. In yetanother embodiment, the human Igλ light chain locus comprises the entirehuman Igλ locus. In another embodiment, the human Igλ light chain locuscomprises human Vλ gene segments and 1 to 7 Jλ-Cλ gene segment pairs,wherein the human Cλ is replaced with syngeneic Cλ.

In one embodiment, the human Igλ light chain locus comprises the humanVλ gene segments, 1 to 7 Jλ gene segments, and a single Cλ gene segment,wherein the gene segments are reconstructed to resemble human Igλconfiguration. In a related embodiment, the method further comprisesconfirming the incorporation of splice sequences into the BAC, andincorporating the splice sequences if not already incorporated into theBAC, prior to the introducing step.

In another embodiment Igλ locus rearranges efficiently, resulting in ahigher representation of Igλ relative to Igκ greater than 40:60. In oneembodiment, the human Vλ gene segments comprise cluster A. A relatedembodiment further comprises the steps of producing a second BACcomprising cluster B human Vλ gene segments; introducing the second BACinto the cell; and replacing endogenous Vλ gene segments. In anotherrelated embodiment, the second BAC further comprises human cluster C Vλgene segments.

One aspect of the invention relates to a method for producing a knock-innon-human mammal that is capable of producing a chimeric Ig heavy chainand a human Ig light chain comprising the steps of breeding a non-humanmammal comprising a chimeric Ig heavy chain locus, wherein the Ig heavychain locus comprises (1) a human VH gene segment and (2) a portion of asyngeneic Ig heavy chain locus comprising all or a part of gene segmentsdownstream of JH, wherein a syngeneic CH1 domain is replaced with ahuman CH1 domain, with a non-human mammal comprising a human Ig lightchain locus; selecting offspring having a genome comprising the chimericIg heavy chain locus and the human Ig light chain locus; furtherbreeding the offspring; and producing offspring having a genomehomozygous for the chimeric heavy chain and human light chain loci.

One embodiment of the invention relates to a method for producing themammal capable of producing a human Ig chain, or a portion thereof, anda human FcR described above comprising the steps of producing a firstBAC comprising a human Ig locus, or a portion thereof; producing asecond BAC comprising a cluster of human FcR genes; introducing thefirst BAC into a homologous recombination competent non-human mammaliancell and replacing all or a portion of an endogenous Ig locus viahomologous recombination; introducing the second BAC into said cell andreplacing all or a portion of an endogenous cluster of FcR genes viahomologous recombination; and generating from the cell a knock-innon-human mammal comprising a genome that encodes a human Ig chain, or aportion thereof, and a human FcR. In a related embodiment, the human Iglocus comprises a portion of an Ig heavy chain locus, said portioncomprising all or a part of the gene segments downstream of JH, suchthat said Ig heavy chain engages a human FcR.

Another aspect of the invention provides a method for producing aknock-in non-human mammal capable of producing a human antibody, whereinthe antibody engages a human FcR, comprising the steps of producing afirst BAC comprising a human Ig heavy chain locus, a second BACcomprising a human Ig light chain locus, and a third BAC comprising acluster of human FcR gene segments; introducing the first, second andthird BACs sequentially into a homologous recombination competentnon-human mammalian cell and replacing an endogenous Ig heavy, Ig light,and FcR gene segment cluster, respectively, via homologousrecombination; and generating from the cell a knock-in non-human mammalcapable of producing a human antibody.

Yet another embodiment relates to a method for producing a knock-innon-human mammal that is capable of producing a human antibody and ahuman FcR comprising the steps of breeding a non-human mammal comprisinga human Ig heavy chain locus, wherein said Ig heavy chain locuscomprises all or a part of the human gene segments downstream of JH,with a non-human mammal comprising a human Ig light chain locus;selecting offspring having a genome comprising the human Ig heavy chainlocus and the human Ig light chain locus; further breeding theoffspring; and producing offspring having a genome homozygous for thehuman heavy and light chain loci.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1C depict the introduction of a chimeric Ig heavy chain viasequential homologous recombination steps. FIG. 1A, FIG. 1B, and FIG. 1Care diagrams that show stage 1, stage 2, and stage 3, respectively, ofselection and screening steps during the homologous recombinationprocess of an IgH locus.

FIGS. 2A-2B depict the introduction of a human Igκ locus via sequentialhomologous recombination steps. FIG. 2A and FIG. 2B are diagrams thatshow stage 1 and stage 2, respectively, of selection and screening stepsduring the homologous recombination of an Igκ locus.

FIGS. 3A-3B depict the introduction of a human Igλ locus via sequentialhomologous recombination steps. FIG. 3A and FIG. 3B are diagrams thatshow stage 1 and stage 2, respectively, of selection and screening stepsduring the homologous recombination process of an Igλ locus.

FIG. 4A is a diagram that depicts the introduction of a first BACcontaining a recognition sequence for a site-specific recombinase into amouse IgH locus via homologous recombination. FIG. 4B is a diagram thatshows the introduction of a second BAC containing a recognition sequencefor a site-specific recombinase into the mouse IgH locus via homologousrecombination. FIG. 4C is a diagram that illustrates the removal of theintervening sequences between the recognition sequences for asite-specific recombinase via introduction of the functionalsite-specific recombinase.

FIG. 5A is a diagram that depicts the introduction of a first BACcontaining a recognition sequence for a site-specific recombinase into amouse Igκ locus via homologous recombination. FIG. 5B is a diagram thatshows the introduction of a second BAC containing a recognition sequencefor a site-specific recombinase into the mouse Igκ locus via homologousrecombination. FIG. 5C is a diagram that illustrates the removal of theintervening sequences between the recognition sequences for asite-specific recombinase via introduction of the functionalsite-specific recombinase.

FIG. 6A is a diagram that depicts the introduction of a first BAC into amouse Igλ locus via homologous recombination. FIG. 6B is a diagram thatshows the introduction of a second BAC into the mouse Igλ locus thathomologously recombines with a portion of the first BAC and a portion ofthe endogenous mouse Igλ locus.

DETAILED DESCRIPTION Overview

The present invention includes knock-in non-human mammals that producechimeric or humanized antibodies, methods of producing such knock-innon-human cells and mammals, and compositions and kits comprising theantibodies produced thereby.

Specifically, embodiments of the invention provide chimeric-antibodyproducing non-human mammals and cells and methods of producing themammals and cells. In antibody producing mammals, the endogenousimmunoglobulin (Ig) V, (D) and J genes are replaced by their humanorthologs using homologous recombination in embryonic stem cells bymethods described herein, such as using BACs carrying large portions ofthe human V, D and J genes and flanked by appropriate homology targetingDNA so as to facilitate high-frequency homologous recombination into theendogenous IgH locus. This can be done in a single replacement in eachIg locus, by sequential (“walking”) replacement, or by replacingportions of the locus followed by removing intervening sequences. A BACcarrying all or part of the IgH locus downstream of JH can be engineeredso that in each constant region gene, the endogenous CH1 domain isreplaced with a human CH1 domain using the ability to make very precisemodifications of DNA carried in BACs in E. coli. Alternatively, a BACcarrying all or part of the IgH locus downstream of JH can be engineeredso that in each constant region gene, the endogenous CH1 domain isreplaced with a human CH1 domain, using the ability to preciselysynthesize and assemble DNAs based on published genome sequences ofhumans and other organisms such as the mouse. Such synthesis andassembly is known in the art and is practiced by commercial entities(e.g., DNA2.0, Menlo Park, Calif.; Blue Heron Biotechnology, Bothell,Wash.).

A benefit of the overall strategy to replace the endogenous Ig loci withcomponents of the human Ig loci so as to produce human V domainsoptimized in vivo during primary and secondary immune responses is thatthe total number of loci altered is reduced compared to availablestrategies. The strategy of the present invention employs 2 or 3 alteredloci, greatly simplifying the breeding process, and thereby providing anopportunity to introduce other useful mutations into the geneticbackground. Such useful mutations may include those that help breakimmune tolerance so as to more efficiently generate antibodies againsthuman antigens that are very highly conserved between species. Theseinclude transgenic over-expression of CD19, knockout of the inhibitoryreceptor FcγRIIb, lpr or other autoimmune prone mutations, knockouts ofthe gene for the antigen, or zinc-finger transgenes engineered tosilence expression of specific genes such as the antigen.

Another benefit of the overall strategy to replace the endogenous Igloci with components of the human Ig loci is that it obviates transrecombination and trans switching that occurs between human Igtransgenes inserted in a chromosomal location outside the endogenousloci. The Ig chain product resulting from such trans recombination andtrans switching events are chimeric for human-endogenous Ig sequencesbut deviate from the product design engineered in the transgene.

Yet another alternative is to incorporate fully human Ig loci includingthe human C regions, in place of the complete endogenous Ig loci andalso replace the cluster of endogenous FcR genes with the orthologouscluster of human FcR genes using a similar BAC-based genetic engineeringin homologous recombination competent cells, such as ES cells. In thisway fully human antibodies can be produced, and during an immuneresponse, these human antibodies can engage the human FcR normally. Suchanimals would also have the benefit of being useful for testing for theactivity of effector function of human therapeutic mAb candidates inanimal models of disease when bred onto the appropriate geneticbackground for the model, i.e., SCID, nu/nu, nod, and lpr mice. Further,the human target gene sequence can replace the endogenous gene using BACtargeting technology in homologous recombination-competent cells, suchas ES cells, providing models for target validation and functionaltesting of the antibody.

Another embodiment incorporates fully human Ig including the human Cregions comprising CH1-hinge-CH2-CH3(-CH4) and the cognate syngeneic,e.g., mouse, membrane and intracellular domains so as to provide nativeintracellular signal transduction and to enable association of the IgHin the B-cell receptor with Igα and Igβ and therein allow murine-typesignaling from the Igα, Igβ and IgG containing B-cell receptor. In yetanother embodiment, the membrane and intracellular domain of the heavychain constant region are from the same or non-cognate mouse heavy chainisotypes. Such engineering of the constant region genes can be readilyaccomplished using methods of the invention as detailed below.

Engineering the chimeric antibodies in this manner prevents thealteration in the V domain conformation resulting from the in vitroswitch from a first C region, particularly a CH1 domain, and optionallya portion of the hinge region, from one species, e.g., mouse, with whichit was evolved during the in vivo immune response to a second C region,particularly a CH1 domain, and optionally a portion of the hinge region,from another species, e.g., human. The antibodies produced by theknock-in animals of the present invention do not exhibit the reductionor loss of activity and potency seen in antibodies from other chimericantibody producing animals when the human V region is appended to ahuman C region to make a fully human antibody, which may be caused byaltered conformation of the VH domain resulting from the changing of theCH1 domain and/or by differences in antigen binding because of changedlength or flexibility of the upper hinge regions (the peptide sequencefrom the end of the CH1 to the first cysteine residue in the hinge thatforms an inter-heavy chain disulfide bond, and which are variable inlength and composition) when switching from mouse to human constantregion (Roux et al., J. Immunology (1997) 159:3372-3382 and referencestherein). The middle hinge region is bounded by the cysteine residuesthat form inter-heavy chain disulfide bonds.

DEFINITIONS

Before describing certain embodiments in detail, it is to be understoodthat this invention is not limited to particular compositions orbiological systems, which can vary. It is also to be understood that theterminology used herein is for the purpose of describing particularillustrative embodiments only, and is not intended to be limiting. Theterms used in this specification generally have their ordinary meaningin the art, within the context of this invention and in the specificcontext where each term is used. Certain terms are discussed below orelsewhere in the specification, to provide additional guidance to thepractitioner in describing the compositions and methods of the inventionand how to make and use them. The scope and meaning of any use of a termwill be apparent from the specific context in which the term is used. Assuch, the definitions set forth herein are intended to provideillustrative guidance in ascertaining particular embodiments of theinvention, without limitation to particular compositions or biologicalsystems. As used in the present disclosure and claims, the singularforms “a,” “an,” and “the” include plural forms unless the contextclearly dictates otherwise.

As used herein, “antibody” or “immunoglobulin” (Ig) refer to proteinmolecules produced by B cells that recognize and bind specific antigensand that may either be membrane bound or secreted. Antibodies may bemonoclonal, in that they are produced by a single clone of B cells andtherefore recognize the same epitope and have the same nucleic acid andamino acid sequence, or polyclonal, in that they are produced bymultiple clones of B cells, recognize one or more epitopes of the sameantigen and typically have different nucleic acid and amino acidsequences.

Antibody, or Ig, molecules are typically comprised of two identicalheavy chains and two identical light chains linked together throughdisulfide bonds. Both heavy chains (IgH) and light chains (IgL) containa variable (V) region or domain and a constant (C) region or domain. Theportion of the IgH locus encoding the V region comprises multiple copiesof variable (V), diversity (D), and joining (J) gene segments. Theportion of the IgL loci, Igκ and Igλ, encoding the V region comprisesmultiple copies of V and J gene segments. The V region encoding portionof the IgH and IgL loci undergo gene rearrangement, e.g., differentcombinations of gene segments arrange to form the IgH and IgL variableregions, to develop diverse antigen specificity in antibodies. Thesecreted form of the IgH C region is made up of three C domains, CH1,CH2, CH3, optionally CH4 (Cμ), and a hinge region. The membrane-boundform of the IgH C region also has membrane and intra-cellular domains.The IgH constant region determines the isotype of the antibody, e.g.IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA and IgE. It will be appreciatedthat non-human mammals encoding multiple Ig isotypes will be able toundergo isotype class switching. There are two types of IgL, Igκ andIgλ.

A “Fab” domain or fragment comprises the N-terminal portion of the IgH,which includes the V region and the CH1 domain of the IgH, and theentire IgL. A “F(ab′)₂” domain comprises the Fab domain and a portion ofthe hinge region, wherein the 2 IgH are linked together via disulfidelinkage in the middle hinge region. Both the Fab and F(ab′)₂ are“antigen-binding fragments.” The C-terminal portion of the IgH,comprising the CH2 and CH3 domains, is the “Fc” domain. The Fc domain isthe portion of the Ig recognized by cell receptors, such as the FcR, andto which the complement-activating protein, C1q, binds. The lower hingeregion, which is encoded in the 5′ portion of the CH2 exon, providesflexibility within the antibody for binding to FcR receptors.

As used herein “chimeric antibody” refers to an antibody translated froma polynucleotide sequence containing both human and non-human mammalpolynucleotide sequences. A “humanized” antibody is one which isproduced by a non-human cell or mammal and comprises human sequences,e.g., a chimeric antibody. Humanized antibodies are less immunogenicafter administration to humans when compared to non-humanized antibodiesprepared from another species. In addition, the humanized antibodies ofthe present invention can be isolated from a knock-in non-human mammalengineered to produce fully human antibody molecules. For example, ahumanized antibody may comprise the human variable region of a chimericantibody appended to a human constant region to produce a fully humanantibody.

As used herein “chimeric Ig chain” refers to an Ig heavy chain or an Iglight chain translated from a polynucleotide sequence containing bothhuman and non-human animal polynucleotide sequences. For example, achimeric Ig heavy chain may comprise human VH, DH, JH, and CH1 genesegments and mouse CH2 and CH3 gene segments.

“Polypeptide,” “peptide” or “protein” are used interchangeably todescribe a chain of amino acids that are linked together by chemicalbonds. A polypeptide or protein may be an IgH, IgL, V domain, C domain,or an antibody.

“Polynucleotide” refers to a chain of nucleic acids that are linkedtogether by chemical bonds. Polynucleotides include, but are not limitedto, DNA, cDNA, RNA, mRNA, and gene sequences and segments.Polynucleotides may be isolated from a living source such as aeukaryotic cell, prokaryotic cell or virus, or may be derived through invitro manipulation by using standard techniques of molecular biology, orby DNA synthesis, or by a combination of a number of techniques.

“Locus” refers to a location on a chromosome that comprises one or moregenes or exons, such as an IgH or Igκ locus, the cis regulatoryelements, and the binding regions to which trans-acting factors bind. Asused herein, “gene” or “gene segment” refers to the polynucleotidesequence encoding a specific polypeptide or portion thereof, such as aVL domain, a CH1 domain, an upper hinge region, or a portion thereof. Asused herein, “gene segment” and “exon” may be used interchangeably andrefer to the polynucleotide encoding a peptide, or a portion thereof. Agene, or gene segment, may further comprise one or more introns,transcriptional control elements, e.g., promoter, enhancers, ornon-coding regions, e.g., cis regulatory elements, e.g., 5′ and/or 3′untranslated regions, poly-adenylation sites.

The term “endogenous” refers to a polynucleotide sequence which occursnaturally within the cell or animal. “Orthologous” refers to apolynucleotide sequence that encodes the corresponding polypeptide inanother species, i.e. a human CH1 domain and a mouse CH1 domain. Theterm “syngeneic” refers to a polynucleotide sequence that is foundwithin the same species that may be introduced into an animal of thatsame species, i.e. a mouse Vκ gene segment introduced into a mouse Igκlocus.

As used herein, the term “homologous” or “homologous sequence” refers toa polynucleotide sequence that has a highly similar sequence, or highpercent identity (e.g. 30%, 40%, 50%, 60%, 70%, 80%, 90% or more), toanother polynucleotide sequence or segment thereof. For example, a DNAconstruct of the invention may comprise a sequence that is homologous toa portion of an endogenous DNA sequence to facilitate recombination atthat specific location. Homologous recombination may take place inprokaryotic and eukaryotic cells.

As used herein, “flanking sequence” or “flanking DNA sequence” refers toa DNA sequence adjacent to the non-endogenous DNA sequence in a DNAconstruct that is homologous to an endogenous DNA sequence or apreviously recombined non-endogenous sequence, or a portion thereof. DNAconstructs of the invention may have one or more flanking sequences,e.g., a flanking sequence on the 3′ and 5′ end of the non-endogenoussequence or a flanking sequence on the 3′ or the 5′ end of thenon-endogenous sequence.

The phrase “homologous recombination-competent cell” refers to a cellthat is capable of homologously recombining DNA fragments that containregions of overlapping homology. Examples of homologousrecombination-competent cells include, but are not limited to, inducedpluripotent stem cells, hematopoietic stem cells, bacteria, yeast,various cell lines and embryonic stem (ES) cells.

“Non-human mammal” refers to an animal other than humans which belongsto the class Mammalia. Examples of non-human mammals include, but arenot limited to, non-human primates, rodents, bovines, ovines, equines,dogs, cats, goats, sheep, dolphins, bats, rabbits, and marsupials.Preferred non-human mammals rely primarily on gene conversion and/orsomatic hypermutation to generate antibody diversity, e.g., mouse,rabbit, pig, sheep, goat, and cow. Particularly preferred non-humanmammals are mice.

The term “knock-in” or “transgenic” refers to a cell or animalcomprising a polynucleotide sequence, e.g., a transgene, derived fromanother species incorporated into its genome. For example, a mouse whichcontains a human VH gene segment integrated into its genome outside theendogenous mouse IgH locus is a transgenic mouse; a mouse which containsa human VH gene segment integrated into its genome replacing anendogenous mouse VH in the endogenous mouse IgH locus is a transgenic ora knock-in mouse. In knock-in cells and non-human mammals, thepolynucleotide sequence derived from another species, may replace thecorresponding, or orthologous, endogenous sequence originally found inthe cell or non-human mammal.

A “humanized” animal, as used herein refers to a non-human animal, e.g.,a mouse that has a composite genetic structure that retains genesequences of the mouse or other non-human animal, in addition to one ormore gene segments and or gene regulatory sequences of the originalgenetic makeup having been replaced with analogous human sequences.

As used herein, the term “vector” refers to a nucleic acid molecule intowhich another nucleic acid fragment can be integrated without loss ofthe vector's ability to replicate. Vectors may originate from a virus, aplasmid or the cell of a higher organism. Vectors are utilized tointroduce foreign or recombinant DNA into a host cell, wherein thevector is replicated.

A polynucleotide agent can be contained in a vector, which canfacilitate manipulation of the polynucleotide, including introduction ofthe polynucleotide into a target cell. The vector can be a cloningvector, which is useful for maintaining the polynucleotide, or can be anexpression vector, which contains, in addition to the polynucleotide,regulatory elements useful for expressing the polynucleotide and, wherethe polynucleotide encodes an RNA, for expressing the encoded RNA in aparticular cell, either for subsequent translation of the RNA into apolypeptide or for subsequent trans regulatory activity by the RNA inthe cell. An expression vector can contain the expression elementsnecessary to achieve, for example, sustained transcription of theencoding polynucleotide, or the regulatory elements can be operativelylinked to the polynucleotide prior to its being cloned into the vector.

An expression vector (or the polynucleotide) generally contains orencodes a promoter sequence, which can provide constitutive or, ifdesired, inducible or tissue specific or developmental stage specificexpression of the encoding polynucleotide, a poly-A recognitionsequence, and a ribosome recognition site or internal ribosome entrysite, or other regulatory elements such as an enhancer, which can betissue specific. The vector also can contain elements required forreplication in a prokaryotic or eukaryotic host system or both, asdesired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, alpha virus and adeno-associated virus vectors, are wellknown and can be purchased from a commercial source (Promega, MadisonWis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or canbe constructed by one skilled in the art (see, for example, Meth.Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly,Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb 25:37-42,1993; Kirshenbaum et al., J. Clin. Invest 92:381-387, 1993; each ofwhich is incorporated herein by reference).

A DNA vector utilized in the methods of the invention can containpositive and negative selection markers. Positive and negative markerscan be genes that when expressed confer drug resistance to cellsexpressing these genes. Suitable selection markers for E. coli caninclude, but are not limited to: Km (Kanamycin resistant gene), tetA(tetracycline resistant gene) and beta-lactamase (ampicillin resistantgene). Suitable selection markers for mammalian cells in culture caninclude, but are not limited to: hyg (hygromycin resistance gene), puro(puromycin resistance gene) and G418 (neomycin resistance gene). Theselection markers also can be metabolic genes that can convert asubstance into a toxic substance. For example, the gene thymidine kinasewhen expressed converts the drug gancyclovir into a toxic product. Thus,treatment of cells with gancylcovir can negatively select for genes thatdo not express thymidine kinase.

In a related aspect, the selection markers can be “screenable markers,”such as green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), GFP-like proteins, and luciferase.

Various types of vectors are available in the art and include, but arenot limited to, bacterial, viral, and yeast vectors. A DNA vector can beany suitable DNA vector, including a plasmid, cosmid, bacteriophage,p1-derived artificial chromosome (PAC), bacterial artificial chromosome(BAC), yeast artificial chromosome (YAC), or mammalian artificialchromosome (MAC). In certain embodiments, the DNA vector is a BAC. Thevarious DNA vectors are selected as appropriate for the size of DNAinserted in the construct. In one embodiment, the DNA constructs arebacterial artificial chromosomes or fragments thereof.

The term “bacterial artificial chromosome” or “BAC” as used hereinrefers to a bacterial DNA vector. BACs, such as those derived from E.coli, may be utilized for introducing, deleting or replacing DNAsequences of non-human mammalian cells or animals via homologousrecombination. E. coli can maintain complex genomic DNA as large as 350kb in the form of BACs (see Shizuya and Kouros-Mehr, Keio J Med. 2001,50(1):26-30), with greater DNA stability than cosmids or yeastartificial chromosomes. In addition, BAC libraries of human DNA genomicDNA have more complete and accurate representation of the human genomethan libraries in cosmids or yeast artificial chromosomes. BACs aredescribed in further detail in U.S. application Ser. Nos. 10/659,034 and61/012,701, which are hereby incorporated by reference in theirentireties.

DNA fragments comprising an Ig locus, or a portion thereof, to beincorporated into the non-human mammal are isolated from the samespecies of non-human mammal prior to humanization of the locus. MultipleBACs containing overlapping fragments of an Ig locus can be humanizedand the overlapping fragments recombine to generate a continuous IgH orIgL locus. The resulting chimeric Ig locus comprises the human genesegments operably linked to the non-human mammal Ig gene segments toproduce a functional Ig locus, wherein the locus is capable ofundergoing gene rearrangement and thereby producing a diversifiedrepertoire of chimeric antibodies.

These processes for recombining BACs and/or of engineering a chimeric Iglocus or fragment thereof requires that a bacterial cell, such as E.coli, be transformed with a BAC containing the host Ig locus or aportion thereof. The BAC containing bacillus is then transformed with arecombination vector comprising the desired human Ig gene segment linkedto flanking homology sequence shared with the BAC containing the host Iglocus or portion thereof. The shared sequence homology mediateshomologous recombination and cross-over between the human Ig genesegment on the recombination vector and the non-human mammal Ig genesegment on the BAC. Detection of homologously recombined BACs mayutilize selectable and/or screenable markers incorporated into thevector. Humanized BACs can be readily isolated from the bacteria andused for producing knock-in non-human cells. Methods of recombining BACsand engineering insertions and deletions within DNA on BACs and methodsfor producing genetically modified mice therefrom are documented. See,e.g., Valenzuela et al. Nature Biotech. (2003) 21:652-659; Testa et al.Nature Biotech. (2003) 21:443-447; and Yang and Seed. Nature Biotech.(2003) 21:447-451.

The first recombination step may be carried out in a strain of E. colithat is deficient for sbcB, sbcC, recB, recC or recD activity and has atemperature sensitive mutation in recA. After the recombination step, arecombined DNA construct is isolated, the construct having the varioussequences and orientations as described.

The regions used for BAC recombineering should be a length that allowsfor homologous recombination. For example, the flanking regions may befrom about 0.1 to 19 kb, and typically from about 1 kb to 15 kb, orabout 2 kb to 10 kb.

The process for recombining BACs to make larger and/or tailored BACscomprising portions of the Ig loci requires that a bacterial cell, suchas E. coli, be transformed with a BAC carrying a first Ig locus, aportion thereof, or some other target sequence. The BAC containing E.coli is then transformed with a recombination vector (e.g., plasmid orBAC) comprising the desired Ig gene segment to be introduced into thetarget DNA, e.g., one or more human VH, DH and/or JH gene segments to bejoined to a region from the mouse IgH locus, both of which vectors havea region of sequence identity. This shared region of identity in thepresence of functional recA in the E. coli mediates cross-over betweenthe Ig gene segment on the recombination vector and the non-human mammalIg gene segment on the BAC. Selection and resolution of homologouslyrecombined BACs may utilize selectable and/or screenable markersincorporated into the vectors. Humanized and chimeric human-mouse BACscan be readily purified from the E. coli and used for producingtransgenic and knock-in non-human cells and animals by introducing theDNA by various methods known in the art and selecting and/or screeningfor either random or targeted integration events.

Alternatively, the DNA fragments containing an Ig locus to beincorporated into the non-human mammal are derived from DNA synthesizedin vitro. The genomes of many organisms have been completely sequenced(e.g., human, chimpanzee, rhesus monkey, mouse, rat, dog, cat, chicken,guinea pig, rabbit, horse, cow) and are publicly available withannotation. In particular but not limited to, the human and mouseimmunoglobulin loci have been studied and characterized for the locationand activity of coding gene segments and non-coding regulatory elements.The sequences of the Ig loci may be manipulated and recombined in silicousing commonly available software for nucleic acid sequence analysis. Insilico recombination may be within the same locus, between two loci fromthe same species, or between two loci from two or more species.Sequences of an Ig locus may also be recombined in silico with thosefrom a non-immunoglobulin locus, either from the same or a differentspecies. Such sequences would include but are not limited to genes forpositive and negative drug selection markers such as G418, hyg, puro andtk, and site-specific recombinase recognition sequences such lox P sitesand its variants and frt sites. After assembling the desired sequence insilico, it may then be synthesized and assembled without errors (Kodumalet al., Proc. Natl. Acad. Sci. (2004) 101:15573-15578). The synthesis,assembly and sequencing of large DNAs are provided on a contractualbasis (e.g., DNA 2.0, Menlo Park, Calif.; Blue Heron Biotechnology,Bothell, Wash.; and Eurogentec, San Diego, Calif.). Such synthetic DNAsequences are carried in vectors such as plasmids and BACs and can betransferred into other vectors such as YACs.

The term “construct” as used herein refers to a sequence of DNAartificially constructed by genetic engineering, recombineering orsynthesis. In one embodiment, the DNA constructs are linearized prior torecombination. In another embodiment, the DNA constructs are notlinearized prior to recombination.

As used herein, “loxP” and “CRE” refer to a site-specific recombinationsystem derived from P1 bacteriophage. loxP sites are 34 nucleotides inlength. When DNA is flanked on either side by a loxP site and exposed toCRE mediated recombination, the intervening DNA is deleted and the twoloxP sites resolve to one. The use of the CRE/lox system, includingvariant-sequence lox sites, for genetic engineering in many species,including mice, is well documented. A similar system, employing frtsites and flp recombinase from S. cerevisiae can be employed to similareffect. As used herein, any implementation of CRE/loxP to mediatedeletional events in mammalian cells in culture can also be mediated bythe flp/frt system.

As used herein the term “immunize,” “immunization,” or “immunizing”refers to exposing the adaptive immune system of an animal to anantigen. The antigen can be introduced using various routes ofadministration, such as injection, inhalation or ingestion. Upon asecond exposure to the same antigen, the adaptive immune response, i.e.T cell and B cell responses, is enhanced.

“Antigen” refers to a peptide, lipid or amino acid which is recognizedby the adaptive immune system. Examples of antigens include, but are notlimited to, bacterial cell wall components, pollen, and rh factor.“Target antigen” refers to an antigen, peptide, lipid, saccharide, oramino acid, which is recognized by the adaptive immune system which ischosen to produce an immune response against a specific infectious agentor endogenous cell. Target antigens include, but are not limited to,bacterial and viral components, tumor-specific antigens, cytokines, cellsurface molecules, etc.

The term “pharmaceutical” or “pharmaceutical drug,” as used hereinrefers to any pharmacological, therapeutic or active biological agentthat may be administered to a subject or patient. In certain embodimentsthe subject is an animal, and preferably a mammal, most preferably ahuman.

The term “pharmaceutically acceptable carrier” refers generally to anymaterial that may accompany the pharmaceutical drug and which does notcause an adverse reaction with the subject's immune system.

The term “administering,” as used herein, refers to any mode oftransferring, delivering, introducing, or transporting a pharmaceuticaldrug or other agent, such as a target antigen, to a subject. Such modesinclude oral administration, topical contact, intravenous,intraperitoneal, intramuscular, intranasal, or subcutaneousadministration.

Knock-in Non-Human Mammals and Cells

Knock-in non-human mammals and cells of the present invention comprisealtered IgH or IgL, or both, loci comprising orthologous human Ig genesegments, which replace the endogenous gene segments. For example, inspecific embodiments a human CH1 domain which replaces a CH1 domain in aspecific endogenous CH gene, e.g., Cμ, Cδ, Cγ, may be an orthologoussequence for each mouse CH region. For each of the endogenous Cγ genes,the replacing CH1 may be an orthologous human CH1 or may be a single Cγfrom human CH genes from human IgG isotype more frequently used intherapeutic mAbs, typically Cγ1, Cγ2 or Cγ4, so as to better facilitatein vivo maturation of human V domain in the context of a more clinicallyrelevant human CH1 domain.

Optionally, the upper hinge sequences of the endogenous C genes may alsobe replaced with the orthologous human C hinge sequences, respectively.Alternatively, the upper and middle hinge sequences of the endogenous Cgenes may also be replaced with the orthologous human C hinge sequences,respectively. If human middle hinge regions are used, the human Cγ4middle hinge sequence may be engineered to contain a proline at residueat position 229 rather than a serine in order to drive inter-heavy chaindimerization via disulfide bonds. The lower hinge regions, part of theCH2 domain, of the endogenous Cγ genes would be left as is to facilitateoptimal binding to endogenous FcγR. This engineering will produce humanheavy chain Fab domain, Fab domain plus upper hinge, or F(ab′)₂ if theupper and middle hinge regions are also converted to human, that will bemore likely to retain optimal characteristics upon conversion to fullyhuman IgG.

BACs comprising the endogenous IgH loci downstream of the J gene clusterand each retained C gene with the human CH1-endogenous CH2-CH3 (and CH4for Cμ) and membrane and intracellular domain exons will be homologouslyrecombined into the endogenous IgH locus in homologousrecombination-competent cells, such as mouse ES cells, either as a firstintroduction step to be followed by replacement of the endogenous V-D-Jgenes with their human orthologs or in the opposite order, introductionof human V, D and J followed by endogenous C genes engineered asdescribed above. The content on the Ig locus on the BAC is notrestricted to only C gene segments on one and V, D and J gene segmentson the other. The BAC with the C gene segments may also contain J genesegments and even D gene segments and even one or more V gene segments.Alternatively, the BAC with V gene segments may contain D gene segmentsand even J gene segments and even one or more C gene segments.

BACs carrying the constant region genes may be engineered in E. coli orsynthesized in vitro prior to introduction into ES cells so as to deleteany unwanted gene segments, such as the Cε and Cα genes. This wouldconstrain the knock-in animals to make Cμ and Cδ for primary immuneresponses and Cγ isotypes for secondary, affinity-matured immuneresponses, from which therapeutic antibody candidates would typically berecovered. The endogenous IgH 3′ LCR may be left unaltered.

A similar replacement strategy would be employed for the endogenous Igκlocus except that the complete human Cκ gene could optionally also beincorporated in the BAC so as to replace the endogenous Cκ locus, thusproducing fully human Igκ chains. The human Cλ locus could also beincorporated in a similar manner.

Yet another aspect of the invention comprises incorporating fully humanIg loci including the human C regions, in place of the completeendogenous Ig loci. In this embodiment, the cluster of endogenous FcRgenes is also replaced with the orthologous cluster of human FcR genesusing similar BAC-based genetic engineering in homologous recombinationcompetent cells, such as mouse ES cells. The cluster of endogenous FcγRgenes can be replaced in the same ES cell in which the human IgH locusor portions thereof have replaced the endogenous locus or in a separateES cell. In the latter instance, mice would be derived from said EScells and bred with mice carrying the engineered Ig locus (loci) so asto produce mice that make human IgG antibodies that bind to human FcγRin place of mouse FcγR genes. In either way fully human antibodies wouldbe produced and during an immune response would be able to engage thehuman FcR receptors normally. Such knock-in animals would also have thebenefit of being useful for testing for the activity and effectorfunction of human therapeutic mAb candidates in models of disease whenbred onto the appropriate genetic background for the model, i.e., SCID,nu/nu, nod, and lpr mice. Further, the human target gene sequence canreplace the endogenous gene using BAC targeting technology in homologousrecombination-competent cells, providing models for target validationand functional testing of the antibody. In this instance, the human CHgenes may be engineered to have cytoplasmic and/or membrane domain genesegments from mouse or other orthologous species to facilitate nativesignal transduction in the B cell.

In addition, another aspect of the invention relates to the design ofthe desired human V region. In particular, an entire human V domainrepertoire or a portion thereof may be incorporated into the genome ofthe cell, or a tailored V domain repertoire may be incorporated. Forexample, in certain embodiments it is preferred to omit V domain genesegments that are missing from some human haplotypes and insteadtailoring the V domain repertoire to be composed of only the functionalV gene segments common across all known human haplotypes. Doing soprovides antibody drug candidates with V domains that are better immunetolerized across all potential patients, thereby preventing theinduction of a dangerous immune response upon administration of theencoded antibody to a subject. One or more V domain gene segments may beincorporated.

In preferred embodiments of the invention, BACs containing the desiredIg loci gene segments are used to incorporate this genetic informationinto the target cell via homologous recombination. The nature of BACengineering in E. coli provides additional opportunities to finelytailor the immunoglobulin loci prior to introduction into competentcells. For example, the human DH cluster can be replaced or supplementedwith D genes from other species, such as from non-human primates,rabbits, rat, hamster etc. BAC libraries and the complete sequence ofthe Ig loci are available for many species. D gene segments within theIgH loci can be defined from publicly available sequence or geneticstructure information, or by testing using appropriate D specific probesor primers. The orthologous D gene clusters or portions thereof can behomologously recombined into the BACs or assembled in silico and thensynthesized, therein replacing or adding to the cluster of human D genesegments. Because of the significant diversification that occurs inmaking the complementarity determining region-3 (CDR3) and because thestructure of the V region is such that the CDR3 is usually solventinaccessible, immunogenicity to the CDR3 sequence is of less concern.Therefore, amino acids encoded by non-human D genes incorporated intothe CDR3 are less likely to be immunogenic. Non-human D genes couldconfer an advantage by producing novel CDR3 structures that would expandthe range of epitope specificities and affinities in a panel ofantigen-specific antibodies, therein broadening the quality ofactivities mediated by a panel of mAbs.

Similarly, the JH gene cluster, i.e., one or more JH gene segments, canbe from a species other than human due to the relative sequenceconservation across mammals. The JH gene segment may be derived from anymammal, e.g., human, non-human primate, rabbit, sheep, rat, hamster, andmouse. In particular embodiments, the JH gene segment is human.

After engineering the Ig loci into homologous recombination-competentcells to replace portions or all of the endogenous Ig loci, geneticallyengineered non-human mammals, such as mice, can be produced bynow-standard methods such as blastocyst microinjection followed bybreeding of chimeric animals, morula aggregation or cloningmethodologies, such as somatic cell nuclear transfer. For example, micewith modified IgH and IgL loci can be bred to produce homozygous IgH andIgL (either Igκ or Igλ). Multi-stage breeding would produce animalshomozygous for modified IgH, Igκ and Igλ loci. Animals heterozygous forIgH and IgL loci, which would produce both fully-mouse and human-mouseantibodies, can also be used to generate antigen-specific human V-mouseC mAbs, though somewhat less efficiently than using homozygous animalsbecause there would also be production of antibody from the activeendogenous Ig loci. Mice heterozygous and homozygous for just onealtered locus, e.g, IgH, could also be useful as a source of human VHdomains (VH-CH1) for antibody display libraries and when mixed with VLdomains from mice heterozygous or homozygous for an altered IgL locus,especially if the mice were immunized (see below). For example, anantibody display library from two separate mice—one with humanVH-CH1-mouse CH2-CH3 and the other with human VK-CK—could be used torecover fully human antibodies using well-established techniques inmolecular biology. Mice heterozygous and homozygous for more than onealtered locus, e.g., IgH and Igκ, would also be useful as a source ofhuman VH and VL domains for antibody display libraries.

Certain embodiments provide a method of producing a knock-in non-humanmammal having a genome encoding human VH and CH1 gene segments and ahuman Ig light chain locus comprising the steps of breeding a non-humanmammal comprising a chimeric Ig heavy chain locus, wherein the Ig heavychain locus comprises the human VH and CH1 gene segments, with anon-human mammal comprising a human Ig light chain locus; selectingoffspring having a genome comprising the chimeric Ig heavy chain locusand the human Ig light chain locus; further breeding the offspring; andproducing offspring having a genome homozygous for the chimeric heavyand human light chain loci. In related embodiments, the genome of themammal also encodes a human JH gene segment.

This genetic engineering strategy can also be applied to engineering ofanimals other than mice so as to express human sequence V regionscoupled with xenogeneic C regions, or completely human antibodies, orsome intermediate thereof. For animals for which there is a current lackof ES cell technology for genetic engineering through blastocystmicroinjection or morula aggregation, the endogenous loci can bemodified in cells amenable to various cloning technologies ordevelopmental reprogramming (e.g., induced pluripotent stem cells, IPS).The increased frequency of homologous recombination provided by the BACtechnology provides the ability to find doubly replaced loci in thecells, and cloned animals derived therefrom would be homozygous for themutation, therein saving time and costs especially when breeding largeanimals with long generation times. Iterative replacements in thecultured cells could provide all the requisite engineering at multipleloci and then direct production of animals using cloning or IPStechnology, without cross-breeding to get the appropriate genotypes. Theability to finely tailor the introduced Ig genes and also finely specifythe sites into which they are introduced provides the ability toengineer enhancements that would provide better function. Engineeredanimals such as goats, bovines, ovines, equines, rabbits, dogs etc.would be a source of fully human polyclonal antibodies.

Furthermore, if the BACs were engineered in E. coli with the DNAcomponents required for chromosome function, e.g., telomeres and acentromere, preferably, but not required, of the recipient species foroptimal function, e.g., mouse telomeres and a mouse centromere, they canbe introduced into the recipient cell by electroporation, microinjectionetc. and would function as artificial chromosomes. These BAC-basedartificial chromosomes also could be used as a foundation for subsequentrounds of homologous recombination for building up larger artificialchromosomes.

The engineered Ig locus or loci described herein on vectors such asplasmids, BACs or YACs can also be used as standard transgenesintroduced via microinjection into the pronucleus of an embryo such asmouse, rabbit, rat, or hamster. Several BACs, YACs, plasmids or anycombination thereof can be co-microinjected and will co-integrate tomake a functional locus. Various methods known in the art can be used toinactivate the endogenous Ig loci and the animals with an engineered Igtransgene bred with those with one or more inactivated endogenous locito derive genotypes expressing antibodies from the transgene and withoutproduction of the complete native immunoglobulin from the inactivatedendogenous loci.

Antibodies

Animals carrying the modified loci can be immunized with target antigensusing various techniques in the art. Target antigens may be selected forthe treatment or prevention of a particular disease or disorder, such asvarious types of cancer, graft versus host disease, cardiovasculardisease and associated disorders, neurological diseases and disorders,autoimmune and inflammatory disorders, and pathogenic infections. Inother embodiments, target antigens may be selected to develop anantibody that would be useful as a diagnostic agent for the detectionone of the above diseases or disorders.

Antigen-specific repertoires can be recovered from immunized mice byhybridoma technology, single-cell RT-PCR for selected B cells, byantibody display technologies, and other methods known in the art. Forexample, to recover mAbs from mouse-derived hybridomas, a humanV-CH1-mouse hinge+CH2+CH3 antibody or a human V-CH1-upper/middlehinge-mouse lower hinge+CH2+CH3 antibody (depending upon the IgH locusengineering) would be secreted into the culture supernatant and can bepurified by means known in the art such as column chromatography usingprotein A, protein G, etc. Such purified antibody can be used forfurther testing and characterization of the antibody to determinepotency in vitro and in vivo, affinity etc.

In addition, since they can be detected with anti-mouse constant regionsecondary agents, the human V-CH1 (upper/middle hinge)-mouse CH2-CH3 mAbmay be useful for immunochemistry assays of human tissues to assesstissue distribution and expression of the target antigen. This featureof the chimeric antibodies of the present invention allows forspecificity confirmation of the mAb over fully human mAbs because ofoccasional challenges in using anti-human constant region secondarydetecting agents against tissues that contain normal human Ig and fromthe binding of human Fc regions to human FcR expressed on cells in sometissues.

The human variable regions of the mAbs can be recovered and sequenced bystandard methods. Either before or after identifying lead candidatemAbs, the genes, either genomic DNA or cDNAs, for the human VH and VLdomains can be recovered by various molecular biology methods, such asRT-PCR, and then appended to DNA encoding the remaining portion of thehuman constant region, therein producing fully human mAb. The DNAsencoding the now fully human VH-CH and human VL-CL would be cloned intosuitable expression vectors known in the art or that can be custom-builtand transfected into mammalian cells, yeast cells such as Pichia, otherfungi etc. to secrete antibody into the culture supernatant. Othermethods of production such as ascites using hybridoma cells in mice,transgenic animals that secrete the antibody into milk or eggs, andtransgenic plants that make antibody in the fruit, roots or leaves canalso be used for expression. The fully human recombinant antibody can bepurified by various methods such as column chromatography using proteinA, protein G etc.

The purified antibody can be lyophilized for storage or formulated intovarious solutions known in the art for solubility and stability andconsistent with safe administration into animals, including humans.Purified recombinant antibody can be used for further characterizationusing in vitro assays for efficacy, affinity, specificity, etc., animalmodels for efficacy, toxicology and pharmacokinetics etc. Further,purified antibody can be administered to humans for clinical purposessuch as therapies and diagnostics for disease.

Various fragments of the human V-CH1-(upper/middle hinge)-endogenousCH2-CH3 mAbs can be isolated by methods including enzymatic cleavage,recombinant technologies, etc. for various purposes including reagents,diagnostics and therapeutics. The cDNA for the human variabledomains+CH1 or just the human variable domains can be isolated from theengineered non-human mammals described above, specifically from RNA fromsecondary lymphoid organs such as spleen and lymph nodes, and the VH andVL cDNAs implemented into various antibody display systems such asphage, ribosome, E. coli, yeast, mammalian etc. The knock-in mammals maybe immunologically naïve or optimally may be immunized against anantigen of choice. By using appropriate PCR primers, such as 5′ in theleader region or framework 1 of the variable domain and 3′ in the humanCH1 of Cγ genes, the somatically matured V regions can be recovered inorder to display solely the affinity matured repertoire. The displayedantibodies can be selected against the target antigen to efficientlyrecover high-affinity antigen-specific fully human Fv or Fabs, and ridof the knock-in mammal of CH2-CH3 domains that would be present if mAbswere recovered directly from the knock-in mammals.

Methods of Use

Purified antibodies of the present invention may be administered to asubject for the treatment or prevention of a particular disease ordisorder, such as various types of cancer, graft versus host disease,cardiovascular disease and associated disorders, neurological diseasesand disorders, autoimmune and inflammatory disorders, allergies, andpathogenic infections. In preferred embodiments, the subject is human.

Antibody compositions are administered to subjects at concentrationsfrom about 0.1 to 100 mg/ml, preferably from about 1 to 10 mg/ml. Anantibody composition may be administered topically, intranasally, or viainjection, e.g., intravenous, intraperitoneal, intramuscular,intraocular, or subcutaneous. A preferred mode of administration isinjection. The administration may occur in a single injection or aninfusion over time, i.e., about 10 minutes to 24 hours, preferably 30minutes to about 6 hours. An effective dosage may be administered onetime or by a series of injections. Repeat dosages may be administeredtwice a day, once a day, once a week, bi-weekly, tri-weekly, once amonth, or once every three months, depending on the pharmacokinetics,pharmacodynamics and clinical indications. Therapy may be continued forextended periods of time, even in the absence of any symptoms.

A purified antibody composition may comprise polyclonal or monoclonalantibodies. An antibody composition may contain antibodies of multipleisotypes or antibodies of a single isotype. An antibody composition maycontain unmodified chimeric antibodies, or the antibodies may have beenmodified in some way, e.g., chemically or enzymatically. An antibodycomposition may contain unmodified human antibodies, or the humanantibodies may have been modified in some way, e.g., chemically orenzymatically. Thus an antibody composition may contain intact Igmolecules or fragments thereof, i.e., Fab, F(ab′)₂, or Fc domains.

Administration of an antibody composition against an infectious agent,alone or in combination with another therapeutic agent, results in theelimination of the infectious agent from the subject. The administrationof an antibody composition reduces the number of infectious organismspresent in the subject 10 to 100 fold and preferably 1,000 fold, andmore than 1,000 fold.

Similarly, administration of an antibody composition against cancercells, alone or in combination with another chemotherapeutic agent,results in the elimination of cancer cells from the subject. Theadministration of an antibody composition reduces the number of cancercells present in the subject 10 to 100 fold and preferably 1,000 fold,and more than 1,000 fold.

In certain aspects of the invention, an antibody may also be utilized tobind and neutralize antigenic molecules, either soluble or cell surfacebound. Such neutralization may enhance clearance of the antigenicmolecule from circulation. Target antigenic molecules for neutralizationinclude, but are not limited to, toxins, endocrine molecules, cytokines,chemokines, complement proteins, bacteria, viruses, fungi, andparasites. Such an antibody may be administered alone or in combinationwith other therapeutic agents including other antibodies, otherbiological drugs, or chemical agents.

It is also contemplated that an antibody of the present invention may beused to enhance or inhibit cell surface receptor signaling. An antibodyspecific for a cell surface receptor may be utilized as a therapeuticagent or a research tool. Examples of cell surface receptors include,but are not limited to, immune cell receptors, adenosine receptors,adrenergic receptors, angiotensin receptors, dopamine and serotoninreceptors, chemokine receptors, cytokine receptors, histamine receptors,etc. Such an antibody may be administered alone or in combination withother therapeutic agents including other antibodies, other biologicaldrugs, or chemical agents.

It is also contemplated that an antibody of the present invention may befurther modified to enhance therapeutic potential. Modifications mayinclude direct- and/or indirect-conjugation to chemicals such aschemotherapeutic agents, radioisotopes, siRNAs, double-stranded RNAs,etc. Other modifications may include Fc regions engineered for eitherincreased or decreased antibody-dependent cellular cytotoxicity, eitherincreased or decreased complement-dependent cytotoxicity, or increasedor decreased circulating half-life.

In other embodiments, an antibody may be used as a diagnostic agent forthe detection one of the above diseases or disorders. A chimericantibody may be detected using a secondary detection agent thatrecognizes a portion of the antibody, such as an Fc or Fab domain. Inthe case of the constant region, the portion recognized may be a CH1,CH2, or a CH3 domain. The Cκ and Cλ domain may also be recognized fordetection. Immunohistochemical assays, such as evaluating tissuedistribution of the target antigen, may take advantage of the chimericnature of an antibody of the present invention. For example, whenevaluating a human tissue sample, the secondary detection agent reagentrecognizes the non-human portion of the Ig molecule, thereby reducingbackground or non-specific binding to human Ig molecules which may bepresent in the tissue sample.

Pharmaceutical Compositions and Kits

The present invention further relates to pharmaceutical compositions andmethods of use. The pharmaceutical compositions of the present inventioninclude an antibody, or fragment thereof, in a pharmaceuticallyacceptable carrier. Pharmaceutical compositions may be administered invivo for the treatment or prevention of a disease or disorder.Furthermore, pharmaceutical compositions comprising an antibody, or afragment thereof, of the present invention may include one or moreagents for use in combination, or may be administered in conjunctionwith one or more agents.

The present invention also provides kits relating to any of theantibodies, or fragment thereof, and/or methods described herein. Kitsof the present invention may include diagnostic or treatment methods. Akit of the present invention may further provide instructions for use ofa composition or antibody and packaging.

A kit of the present invention may include devices, reagents, containersor other components. Furthermore, a kit of the present invention mayalso require the use of an apparatus, instrument or device, including acomputer.

EXAMPLES

The following examples are provided as further illustrations and notlimitations of the present invention. The teachings of all references,patents and published applications cited throughout this application, aswell as the Figures are hereby incorporated by reference.

Example 1 Design of BACs in E. Coli

As described in US Patent Application Publication No. 2004/0128703, themanipulation of BACs in E. coli provides a powerful tool for finetailoring of the genomic DNA carried in the BACs. For example, toreplace mouse CH1 with human CH1 in one or more of the mouse constantregion genes, e.g., Cγ1 or all mouse Cγ genes or mouse Cμ, Cδ and allthe mouse Cγ genes, a modified mouse BAC is made in E. coli and thenused for homologous recombination in ES cells. For example, in thetargeting BAC, the mouse CH1 exons of at least one and up to all of theCγ genes are replaced by the human CH1 exons. This replacement issimilarly performed in E. coli using a homologous recombination method.The resulting modified BAC has a germline-configured segment carryingthe human D region, the human J region, and downstream mouse Cμ region,Cδ region, Cγ3 region (mouse CH1 of γ3 is replaced by human CH1),modified Cγ1 region (mouse CH1 of γ1 is replaced by human CH1), modifiedCγ2B region (mouse CH1 of γ2B is replaced by human CH1), and modifiedCγ2C (mouse CH1 of γ2C is replaced by human CH1). This strategy can alsobe used to modify the CH1 exons of Cμ and Cδ genes. Other precisereplacements such as the upper and middle hinge region coding sequencewith human for mouse can also be made. Further finely tailored changesincluding as small as single codon and single nucleotide changes can beengineered into the replacing DNA.

Example 2 Homologous Recombination of BACs in E. Coli

A BAC vector is based on the F-factor found in E. coli. The F-factor andthe BAC vector derived from it are maintained as low copy plasmids,generally found as one or two copies per cell depending upon its lifecycle. Both F-factor and BAC vector show the fi⁺ phenotype, whichexcludes an additional copy of the plasmid in the cell. By thismechanism, when E. coli already carries and maintains one BAC, and thenan additional BAC is introduced into the E. coli, the cell maintainsonly one BAC, either the BAC previously existing in the cell or theexternal BAC newly introduced. This feature is extremely useful forselectively isolating BACs homologously recombined as described below.

The homologous recombination in E. coli requires the functional RecAgene product. In this example, the RecA gene has a temperature-sensitivemutation so that the RecA protein is only functional when the incubationtemperature is below 37° C. When the incubation temperature is above 37°C., the Rec A protein is non-functional or has greatly reducedrecombination activity. This temperature sensitive recombination allowsmanipulation of RecA function in E. coli so as to activate conditionalhomologous recombination only when it is desired. It is also possible toobtain, select or engineer cold-sensitive mutations of Rec A proteinsuch that the protein is only functional above a certain temperature,e.g., 37° C. In that condition, the E. coli would be grown at a lowertemperature, albeit with a slower generation time, and recombinationwould be triggered by incubating at above 37° C. for a short period oftime to allow only a short interval of recombination.

Homologous recombination in E. coli is carried out by providingoverlapping DNA substrates that are found in two circular BACs. Thefirst BAC (BAC1) carries the contiguous segments from A through E, andthe second BAC (BAC2) carries the contiguous segments from E through I.The segment E carried by both BACs is the overlapping segment where theDNA crossover occurs, and as a result it produces a recombinant thatcarries the contiguous segments from A through I.

BAC1 described above is the one already present in the cell, and whenBAC2 is introduced into the cell, either BAC1 or BAC2 can exist in thecell, not both BACs. Upon electroporation of BAC2 into the cell, thetemperature would be lowered below 37° C. so as to permit conditionalRecA activity, therein mediating homologous recombination. If BAC1 andBAC2 have a selectable marker each and the markers are distinctivelydifferent, for example, BAC1 carries Kan (a gene conferring kanamycinresistance) and BAC2 carries Amp (a gene giving Ampicilin resistance),only the recombinant BAC grows in the presence of both antibiotics Kanand Amp.

Since there are two E gene segments at the separate region of therecombinant BAC, the E segment flanked by two vectors must be removed byone of two ways, one is by homologous recombination at either thevectors or the E region, and the other is carried out by loxP sitespecific recombination by CRE recombinase. The resolved BAC has now thecontiguous stretch from A through I with single copy of E.

Example 3 Homologous Recombination in E. Coli

According to the procedure detailed above, BAC1 and BAC2 undergorecombination in E. coli. BAC1 and BAC2 have 156,427 bp and 122,500 bp,respectively, and they overlap by 42,363 bp. The size of the resultingBAC after recombination and resolution is 236,564 bp. BAC2 containing anAmp gene in the BAC vector or in the appropriate area of the BAC isintroduced into an E. coli bacillus carrying BAC1 having a Kan gene inthe BAC vector or in the appropriate area of the BAC.

The introduction of a BAC to E. coli cell is typically done byelectroporation. Prior to electroporation, the cells are maintained at40° C., a non-permissive temperature for recombination, and afterelectroporation the cells are incubated at 30° C., a temperaturepermissive for recombination. During the incubation, homologousrecombination occurs and cells express enzymes necessary to becomeresistant to both antibiotics. The incubation period is about 45 to 90minutes. Then the cells are spread on the media plates containing bothantibiotics and the plates are incubated at 40° C. to prevent furtherhomologous recombination. The majority of colony isolates growing on themedia plates have the recombined BAC that has predicted size. This canbe confirmed by pulsed field gel electrophoresis analysis. Integrity ofthe recombined DNA is confirmed with restriction digests usingrare-cutting and infrequent cutting restriction enzyme digests analyzedby pulsed field gel electrophoresis.

Example 4 Design of a BAC to Replace Endogenous CH1 with Human CH1

In this example, two BACs (173, 869 bp and 102,413 bp) overlap by 6,116bp recombine to produce a 270,165 bp BAC. To replace mouse CH1 in themouse BAC with human CH1, an appropriate DNA construct is made. Theconstruct is made by chemical synthesis, PCR, conventional cloningtechniques or a combination of these methods. The construct has a humanCH1 sequence flanked by mouse sequence arms that, in the mouse genome,flank the mouse CH1 region as well.

The construct is recombined in E. coli with the original mouse BAC atone or the other side of the homologous flanking mouse DNA sequences,and after repeated recombination and resolution by Cre recombinase, achimeric BAC having human CH1 replacing mouse CH1 is made. By repeatingthis process, all of the mouse CH1 regions of the Cγ genes are replacedwith human CH1 regions, producing a modified BAC carrying all of the Cγgenes having human CH1 in place of the mouse CH1.

Similarly, the mouse CH1 domains of Cμ, Cδ can be replaced by thecorresponding human CH1 domains. If desired, Cα and Cε can also bemodified in this manner. Similar replacement(s) can also be performedwith the upper and/or middle hinge segments of the C gene(s). Ifincorporating a human middle hinge region, the human Cγ4 middle hingeencoding DNA sequence can be engineered, such as by chemical synthesis,with a codon encoding proline replacing the codon encoding serine 229 toeffect interchain rather than intrachain disulfide bind formation tostabilize the IgG4 dimer.

Example 5 Introduction of BACs into Cells

In preparation for introduction into ES cells, mammalian expressioncassettes for selectable markers and for screenable markers can berecombined onto the BACs. Such cassettes carry genes with requiredregulatory elements such as promoters, enhancers and poly-adenylationsites for expression of the genes in mammalian cells, such as mouse EScells. The genes on the cassette can be selectable markers such asdrug-resistance and drug-sensitivity genes for drugs such as G418,hygromycin, puromycin, gancyclovir (thymidine kinase), hypoxanthinephosphoribosyl transferase etc. and screenable markers such asgreen-fluorescent protein (GFP), red-fluorescent protein (RFP),luciferase etc. Such markers are used to select and screen for cellsinto which the BAC has been introduced and homologously recombined.

For introduction into ES cells, BAC DNA is purified from E. coli and theE. coli genomic DNA by methods known in the art such as the alkalinelysis method, commercial DNA purification kits, CsCl density gradient,sucrose gradient, or agarose gel electrophoresis, which may be followedby treatment with agarase. To linearize the purified DNA, it is thendigested by NotI. The two NotI sites flank the cloning site on the BACvector and thus NotI digestion separates the insert from the vector. Theinsert DNA is free from the vector DNA, which has a loxP site, exceptthe small region between the cloning site and NotI site. Thus, the DNAto be transfected into the mammalian cell does not carry a loxP siteunless one is purposely engineered into the DNA to be transfected intothe mammalian cell. Although NotI site is extremely rare on human andmouse immunoglobulin genomic DNA, if the BAC DNA construct contains oneor more NotI sites, sites for other rare restriction enzymes such asAscI, AsiSI, FseI, PacI, PmeI, SbfI, and SwaI, homing endonucleases suchas I-CeuI, I-SceI, PI-PspI, PI-SceI, or lambda terminase will beintroduced into the junction area between the insert and the vector.This can be accomplished by transposon, homologous recombination, andother cloning methods. The linearized DNA, typically 0.1-10 μg of DNAdepending upon the size, are introduced into the mammalian cells, suchas ES cells, by methods known in the art such as transfection,lipofection, electroporation, Ca-precipitation or direct nuclearmicroinjection.

Example 6 Engineering of Ig Loci Via Incorporation of Large BACs ViaSequential Replacement in Embryonic Stem Cells

Homologous recombination in E. coli to construct larger BACs isdescribed in U.S. Patent Application Publication No. 2004/0128703. Suchmethods can be used to make BACs with larger inserts of DNA than isrepresented by the average size of inserts of currently available BAClibraries. Such larger inserts can comprise DNA representing the humanIg loci such IgH, Igκ and Igλ. The DNA inserts can also comprise DNArepresenting the endogenous Ig loci including some or all of the DNArepresenting the constant region genes, which also may carrymodifications designed into the DNA.

As an example, using such longer BACs, the region containing the humanIgH V, D and J gene segments is covered by only 3 to 4 separate BACs,each of which would be approximately ˜300 kb in size (FIG. 1). Theregion containing the mouse IgH constant region gene segments fromdownstream of the JH gene segment cluster through just upstream of themouse 3′ locus control region can be covered in a single BAC of 170-180kb. These BACs also carry overlapping segments to provide homology withthe subsequent BAC for homologous recombination in cells such as EScells. Using these longer BACs, the V, D and J gene segments of themouse IgH are sequentially replaced with the corresponding humansequences and the constant region gene segments of the mouse IgH arereplaced with engineered human-mouse constant region gene segments. Thesequential replacements are substantially complete to the desiredamount.

The first BAC to be introduced into ES cells may be comprised of humanIg DNA flanked on either side by 1 kb to 10 kb to 100 kb or more ofmouse DNA from the corresponding endogenous mouse genome in the ES cell.The first BAC then replaces a portion of the endogenous mouse genome byhomologous recombination in ES cells, replacing the endogenous mouse DNAbetween the two flanking DNAs, which are the targeting sites, with thehuman DNA engineered between the flanking DNAs on the BAC. For example,by constructing in E. coli a BAC that contains a known size in kilobasesof human IgH DNA that contains human variable regions, the DH genecluster and the JH cluster, flanked on the 3′ end by mouse DNAcorresponding to the region 3′ of the mouse JH locus and flanked 5′ bymouse DNA corresponding to the region the same known size in kilobases5′ of the mouse JH cluster, and introducing the purified BAC into mouseES cells to allow for homologous recombination, the corresponding mouseVH, DH and JH genes would be replaced by the orthologous human DNA.

The flanking mouse DNAs could also be further away, e.g., the 5′homology could be upstream of the most 5′ endogenous VH gene so thatupon homologous recombination, the entire mouse VH-DH-JH region would bereplaced by the human VH-DH-JH on the BAC. In other words, the length ofthe region of the endogenous DNA to be replaced is determined by thedistance between the two flanking mouse segments on the BAC. Thedistance is not the actual length between the flanking mouse segments inthe BAC; rather it is the distance between the flanking mouse segmentsin the endogenous mouse chromosome. This distance may be calculated fromthe available genomic databases, such as UCSC Genomic Bioinformatics,NCBI and others known in the art.

A second, and any subsequent, BAC would have two segments flanking theDNA to be introduced. For the two flanking DNAs, one is comprised ofhuman DNA that corresponds to all or a portion of the human DNAintroduced into the cell genome in the first replacement and the otheris mouse DNA corresponding to endogenous DNA upstream (or downstream asthe case may be) of the region to be replaced in the secondintroduction.

Upon introduction into a homologous recombination-competent cell such asa mouse ES cell into which a first BAC DNA has replaced a portion of theendogenous locus, e.g., into the mouse IgH locus, one crossover wouldoccur between the human flanking sequence of the BAC and the humansequence in the modified mouse chromosome, and the other between themouse sequence of the BAC and the corresponding mouse region of saidchromosome (see FIG. 1B and FIG. 10). Put another way, in the secondBAC, the human flanking sequence includes a homologous segment to theend portion of the human DNA sequence of the first BAC previouslyintegrated into the chromosome.

In this way, when they are joined by homologous recombination in EScells, the joined segments become a contiguous segment. Such a segmentmay be germline-configured as it is naturally found in humans or becauseof purposely engineered insertions, deletions or rearrangements may havea configuration different from germline. The mouse flanking sequence inthe second BAC corresponds to the mouse endogenous chromosomal DNA thatis a specified, desired distance away from the newly introduced humansequence in the mouse chromosome. The distance between the two mousesegments are long enough so that the replacement of the mouse endogenousIgH is completed as planned after repeated homologous recombination.

For instance, using BACs with suitable human DNA inserts of the VH, DH,and JH loci and flanked with suitably located targeting DNAs, it wouldbe possible to replace all of the endogenous mouse VH, DH and JH geneswith their corresponding human counterparts with 2-3 sequentialreplacements with BACs suitably engineered in E. coli as outlined aboveto facilitate homologous recombination in mammalian cells, including EScells, and therein leaving the human V-D-J genes operably linked todownstream mouse constant region loci. In a third, fourth or fifthreplacement, using a BAC with a suitable human DNA insert carrying humanCμ, Cδ and Cγ genes and optionally Cα, Cε and the human 3′ locus controlregion (LCR), and flanked by human and mouse DNAs corresponding tointroduced and endogenous DNA, respectively, the endogenous mouse Cregions can be replaced with their human counterparts.

Alternatively, the genomic DNA comprising the constant region genes maybe of mouse origin and may be engineered with desired modifications suchas replacing the CH1 domain of all or selected mouse constant regionswith human CH1 DNA, and/or upper and the optional middle hinge regionsof some or all of the mouse C genes with corresponding human genesequences, all flanked by appropriate targeting DNA as outlined above.Further, some of the mouse C regions, e.g., Cε and/or Cα and/or one andup to all of the Cγ gene segments, can be deleted such that theendogenous mouse 3′ LCR would be in closer than germline proximity tothe most 3′ constant region on the BAC, and upon homologousrecombination into the genome, effecting deletion of the endogenous Cgene segments absent from the targeting BAC.

The order of introduction of the BACs and sequential replacements can beeither distal to proximal or proximal to distal.

Example 7 Engineering of Ig Loci Via Incorporation of Large BACs and Useof Site-Specific Recombinases in Embryonic Stem Cells

Alternatively, sites for site-specific recombinases, such as loxP/CRE orfrt/flp, can be employed to facilitate engineering of the Ig loci byintroducing site-specific recombinase recognition sequences into DNAs tobe introduced on the homologous recombination vectors, and then when thesite-specific recombinase is expressed or introduced, recombination willoccur between the sequences, therein deleting the intervening sequences.In this way two or more non-overlapping BACs are incorporated into theendogenous Ig locus via homologous recombination. The 5′ BAC contains aloxP or frt recognition sequence in its 3′ region while the 3′ BACcontains a loxP or frt site in its 5′ region, and the remainingendogenous sequence between the newly introduce sequences is removed viasite-specific recombination.

The BACs can be engineered to introduce loxP or frt sites that willflank the intervening sequences. Subsequently, expression of CRE or flprecombinase, respectively, in either the cells or the geneticallyengineered organism derived therefrom will trigger site-specificrecombination between the loxP and frt sites, thereby deleting theintervening sequences (FIGS. 4 and 5).

After homologously recombined clones incorporating the second BACtargeting vector in the precise location have been identified, includingperformance of assay, e.g., fluorescent in situ hybridization with DNAprobes from the first and second BAC, to confirm cis integration of thesecond BAC on the chromosome carrying the first BAC integrant, the firstand second BACs are separated by an amount of intervening endogenous DNAfrom the mouse locus. The amount and content of this interveningendogenous DNA is determined by the location of the 3′ flanking DNA onthe first BAC and the 5′ flanking DNA on the second BAC. This remainingintervening portion of the mouse sequence contained between the loxP orefrt sites that were introduced by homologous recombination is removed byCRE recombinase or flp recombinase. Either CRE recombinase or flprecombinase can be transiently expressed in clones that have bothcorrectly targeted BAC inserts. CRE recombinase or flp recombinase actsefficiently and precisely upon loxP site or flp sites, respectively,therein deleting the intervening DNA between said sites. Confirmation ofdeletion and precise joining of the two BACs, 3′ of the first BAC joinedto the 5′ of the second BAC, can be detected by Southern blots asdescribed herein.

In yet another alternative, a lox P site and Cre recombinase can be usedto selectively introduce a lox-site carrying exogenous DNA into a lox-Psite already incorporated into the engineered Ig loci. In this way,additional DNA content can be introduced into the engineered loci.

Example 8 Sequential Replacement of Endogenous DNA from the 5′ Direction

The direction of the replacement in homologous recombination-competentcells, such as ES cells, may be performed either from the 5′ end or 3′end of the transcriptional direction. However, BAC modification shouldbe done according to the configuration of the homology requirement forhomologous recombination in competent cells.

For example, in the 5′ end direction, the first BAC to be used has thetelomere side of IgH V gene segments of the human sequence, flanked oneither side by endogenous mouse DNA for targeting into the mouse IgHlocus. The final BAC to be used in the iterative replacement process isa BAC modified as described above having human CH1 domains replacingmouse CH1 domains in some or all of the endogenous mouse constantregions. The DNA upstream of the mouse C region germline configured DNA(with the exception of CH1 domain replacement(s) and optionally upperand optionally also the middle hinge replacements) would be human DNAcorresponding to a portion already integrated into the modified IgHlocus and the downstream DNA would be mouse sequence 3′ of the most 3′mouse Cγ on the replacing DNA to effect deletion of Cα and Cε and any Cγgene segments but to leave unaffected the content of the mouse 3′ LCR.As noted above, the flanking DNAs may range in size from 1 kb to 10 kbto 100 kb to larger.

Example 9 Sequential Replacement of Endogenous DNA from the 3′ Direction

In the 3′ direction, the first BAC is a modified BAC based on the lastBAC for the 5′ directional replacement. Furthermore, the first BAC hasan additional 1 kb to 10 kb to 100 kb or greater mouse segment at theother side of the human sequence. For example, the 1 kb to 10 to 100 orgreater kb segment starts from around the 5′ end of the D region towardthe telomere of the mouse chromosome. The last BAC is a modified BAC ofthe first BAC used for the replacement from 5′ direction. Themodification is an addition of 1 kb to 10 to 100 or greater kb of themouse segment to the end of the human sequence. The 1 kb to 10 to 100 orgreater kb region starts from the end of the first IgH V gene segmenttoward the outside of IgH.

Example 10 Homologous Recombination of the Igκ Locus

A replacement strategy as outlined above can also be performed on an Igκlocus endogenous in a cell chromosome, for example, in a mouse ES cell.To replace the endogenous mouse Vκ genes with the corresponding human Vκgene content, only two iterative replacements may be required. This isbecause the human Vκ gene content is redundant, with the human Vκ genesbeing represented about 2 times, with a proximal cluster oriented in thesame 5′-3′ orientation as the Jκ and Cκ gene and this cluster duplicatedin a distal, inverted orientation. This inverted, duplicated clusterrepresents only about 10% of the expressed Vκ repertoire in humans.Further, this distal, inverted duplication is missing from about 10% ofhumans. Consequently, as little as two overlapping BACs could comprisethe unique human Vκ repertoire plus human Jκ genes and the human Cκ geneas shown in FIG. 2.

Alternatively, two non-overlapping BACs are homologously recombined intothe Igκ locus, and each BAC introduces a site specific recombinaserecognition sequence, such as loxP or frt. The 5′ BAC contains, forexample, a site-specific recombination recognition site in its 3′region, and the 3′ BAC contains a site-specific recombinationrecognition site in its 5′ region. Upon expression of the site-specificrecombinase, the intervening mouse Igκ sequences between the twosite-specific recombination recognition sites are deleted, therebyjoining the sequences introduced by the BACs (FIG. 5).

Example 11 Homologous Recombination of the Igλ Locus

A replacement strategy as outlined above can also be performed on theIgλ locus endogenous in a cell chromosome, such as in a mouse ES cell.To replace the endogenous mouse Vλ genes with the corresponding human Vλgenes, only two iterative replacements may be required (FIG. 3).However, because the genomic organization of the Igλ locus is differentin mouse versus humans, an alternative engineering strategy must bepursued to implant the human Igλ Vλ-Jλ genes. In mice, the Igλ locusconsists of two separate but linked gene clusters, each composed of oneor two Vλ genes upstream of two paired Jλ-Cλ gene sets. The mostupstream Vλ only very rarely rearranges to the most downstream Jλ-Cλpair. In humans, there are approximately 30 functional Vλ genes upstreamof 7 Jλ-Cλ clusters.

Furthermore, leaving the mouse constant regions, specifically the mouseIgλ 3′ LCRs, Eλ2-4 and/or Eλ3-1, intact and unmodified would likely leadto an engineered locus that would fail to express at the 60/40 κ:λ ratioof humans and rather would more likely express at the 95/5 κ:λ ratio ofmice because of mutations, probably causing defects in NF-κB binding,that are present in the mouse Igλ 3′ LCRs (see, e.g., Combriato andKlobeck, J. Immunol. 168:1259-1266). Because the human Igλ locuscontributes a substantial amount of diversity to the total human lightchain repertoire it is beneficial to appropriately engineer the humanIgλ BAC to recapitulate the genuine human immune response so as toincrease the efficiency and probability of generating therapeuticallyuseful mAbs against a broad range of antigen targets.

Engineered BACs replace the endogenous mouse Igλ locus with the humancounterpart and restore a more fully functional Igλ 3′ LCR downstream ofthe constant regions (FIG. 6). Several alternative strategies can beemployed. The entire mouse Igλ locus can be replaced by the human locusby sequentially targeting a series of BACs that overlap across the humanIgλ locus including the fully functional human Igλ 3′ LCR into the mouseIgλ locus, sequentially replacing all of it, including the 3′ LCRs.Alternatively, overlapping BACs can be engineered to contain all, or aportion of, the human Vλ genes and the 1-7 Jλ-Cλ pairs but with mouse Cλgenes replacing the human Cλ genes so that chimeric human Vλ-mouse CλIgλ chains are produced by the mouse. Alternatively, the human Igλ locuscan be re-constructed on overlapping BACs to resemble the configurationof the human Igκ locus, with the complete set, or subset, of human Vλgenes, a cluster of 1-7 Jλ genes and a single selected human, or mouse,Cλ gene followed by the human 3′ LCR. The inclusion of appropriatesplice donor and splice acceptor sequences in the Jλ cluster and in theCλ gene is confirmed, and if required, engineered into the BAC(s) in E.coli or during the chemical synthesis and assembly, prior tointroduction into ES cells. This reconfigured locus rearranges andsplices appropriately. However, it is possible that such a constructcould rearrange more efficiently than in the germline configured humanIgλ locus, therein leading to a proportionally higher representation ofIgλ relative to Igκ in the light chain repertoire than is seen inhumans.

Other configurations of the human locus and introductions into the mouseIgλ locus so as to produce human Igλ V regions conjoined with eitherhuman or mouse Cλ genes can be readily designed and engineered. Eitherthe human Igλ 3′LCR region with functional NFκB binding sites or themouse Igλ 3′LCR control with introduced mutations, e.g., bysite-directed mutagenesis in vitro, to restore NFκB binding (see, e.g.,Combriato and Klobeck, J. Immunol. 168:1259-1266), or a functionalsub-portion of either one, such as DNAse I hypersensitive site 3 of thehuman Igλ 3′LCR, is included downstream of the final Cλ gene in acluster in all constructs. Alternatively, a functional Igλ 3′LCR orfunctional sub-portion thereof from another species, e.g., rat,non-human primate, could be included. Alternatively, a functional Igκ 3′LCR from human, mouse or other species could be used.

Unless specifically deleted from or the sequence for which is notincluded in the chemically synthesized and assembled human DNA, theprocess of introducing the human Vλ repertoire will also introduce thegenes for human surrogate light chain (SLC), which is within the humanVλ gene array in the germline DNA. Mouse SLC genes are linked to, buthundreds of kilobases separate from, the Igλ locus and would beunperturbed in this strategy so transgenic mice would co-express humanand mouse SLC.

The human Vλ repertoire can be grouped into three clusters: A, B and C.The A cluster, most proximate to the J-C pairs, is the most frequentlyused, followed by the B and then the C cluster. One, two or three ofthese Vλ clusters may be incorporated. The strategy herein allows forengineering any or all or a portion thereof of the human Vλ clustersinto the mouse genome, and replacing the endogenous mouse locus.

Example 12 Selection of Non-Human Mammalian Cells Following HomologousRecombination

To identify mammalian cells, such ES cells, that are the result ofhomologous recombination, a series of selection and screening proceduresfollowed by molecular analyses are employed (FIGS. 1, 2 and 3). First,the cells are grown in the presence of a drug for which at least onedrug-resistance gene is represented on the introduced BAC so as toselect for cells that are stably carrying the BAC. The BAC mayoptionally carry a negative-selection marker such as thymidine kinase atthe outside terminus of one or both of the flanking targeting regions toselect against random integrants. Alternatively, clones positive for onedrug resistance marker could be picked and duplicate plates made, one totest for drug resistance and one to test for drug sensitivity.Optionally, the BAC would also carry a screenable marker such as GFP orRFP approximately adjacent to the selectable marker. GFP⁺ or RFP⁺ clonescould be detected by FACS or fluorescence microscopy. Both positiveselectable and screenable markers are internal to the flanking targetingDNA so as to be stably integrated into the genome along with thereplacing DNA.

To confirm homologous recombination on selected (drug resistant) and,optionally, screened (e.g., GFP⁺) clones, genomic DNA is recovered fromisolated clones and restriction fragment length polymorphism (RFLP)analysis performed by a technique such as Southern blotting with a DNAprobe from the endogenous loci, said probe mapping outside the replacedregion. RFLP analysis shows allelic differences between the two alleles,the endogenous DNA and incoming DNA, when the homologous recombinationoccurs via introduction of a novel restriction site in the replacingDNA. Because flanking DNA arms >10 kb in size may generate RFLPs thatare large and difficult to resolve by standard agarose gelelectrophoresis, low percentage agarose gels may be used or CHEF gelelectrophoresis may be used. Flanking DNA arms of about 10 kb or less insize generate RFLPs that can be resolved by standard agarose gelelectrophoresis. Alternatively, a restriction site may be purposelyengineered into the replacing DNA on the BAC during replacement vectorconstruction so as to engineer a conveniently sized fragment spanningthe junction of the introduced DNA and the endogenous DNA uponrestriction digest, and encompassing the designated probe sequence. Inaddition to RFLP analysis, cis integration may be screened usingfluorescence in situ hybridization (FISH) to confirm the location of theintroduced DNA according to methods known in the art.

For subsequent engineering, different selectable and/or screenablemarkers are just internal to one flanking arm while the oppositeflanking arm for homologous recombination, which overlaps with theflanking arm carrying the selectable and/or screenable markers used intargeting the BAC1, carries no markers, such that the homologousrecombination event deletes the markers introduced in targeting BAC1 andintroduces a new selectable and/or screenable marker at the opposite end(internal from the opposite flanking arm). For example, fluorescentmarkers alternate between GFP and RFP after each round of homologousrecombination occurs such that round 1 introduces GFP and round 2deletes GFP and introduces RFP. If random insertion occurs, bothfluorescent markers exist in the ES cells. A flow cytometer with cellsorting capability can be utilized to sort and retain cells based on thepresence of signals from one fluorescent protein and the absence ofsignal from another. Drug resistance markers can be used similarly,e.g., using a pair of positive and negative selection markers (FIGS. 6Aand 6B).

Through standard advanced planning it should be possible to replaceendogenous DNA with human DNA across megabase-sized loci throughiterative rounds of homologous recombination using only 2 differentpairs of combinations of one selectable marker and one screenablemarker. However, three or more sets each of selectable and screenablemarkers could also be used.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method for obtaining an antibody that binds to a target antigen,which method comprises: a. immunizing a transgenic mouse with a targetantigen, wherein the transgenic mouse has a genome comprising animmunoglobulin (Ig) heavy chain locus comprising a transgene encoding achimeric Ig heavy chain, the transgene comprising a portion of a mouseIg heavy chain locus, the portion comprising all or part of constantregion genes encoding a chimeric Ig heavy chain, the transgenecomprising a DNA fragment comprising in operable linkage from 5′ to 3′ aDNA sequence of a human variable heavy (VH) segment gene operably linkedto a constant region gene comprising a human CH1, human C_(upper hinge),mouse non-human CH2 and mouse non-human CH3 exons, wherein the human CH1exon is selected from the group consisting of a human Cμ, a human Cδ, ahuman Cγ1, a human Cγ2, a human Cγ4, a human Cα, and a human Cε, therebythe transgenic mouse comprises a genome encoding the chimeric Ig heavychain; and b. recovering antibodies comprising the chimeric Ig heavychain that bind to the predetermined antigen.
 2. The method according toclaim 1, wherein recovering comprises using hybridomas.
 3. The methodaccording to claim 2, further comprising isolating a polynucleotideencoding a variable region of the chimeric Ig heavy chain.
 4. The methodof claim 3, further comprising recombinantly making a variable regionpolypeptide encoded by the isolated polynucleotide.
 5. The methodaccording to claim 4, wherein recombinantly making comprises an antibodydisplay system, a eukaryotic expression system, a prokaryotic expressionsystem, or an in vitro translation system.
 6. The method according toclaim 3, wherein the isolated polynucleotide is sequenced and asynthetic nucleic acid molecule is produced from the sequence of theisolated polynucleotide.
 7. The method according to claim 6, wherein thesynthetic nucleic acid molecule comprises the human VH, the human CH1, ahuman CH2 and a human CH3.
 8. The method according to claim 1, whereinrecovering comprises selecting B cells that express the antibodiescomprising the chimeric Ig heavy chain that bind to the predeterminedantigen.
 9. The method according to claim 8, further comprisingisolating a polynucleotide encoding a variable region of the chimeric Igheavy chain from the B cells.
 10. The method according to claim 9,wherein the isolating comprises single-cell RT-PCR.
 11. The method ofclaim 9, further comprising recombinantly making a variable regionpolypeptide encoded by the isolated polynucleotide.
 12. The methodaccording to claim 11, wherein recombinantly making comprises anantibody display system, a eukaryotic expression system, a prokaryoticexpression system, or an in vitro translation system.
 13. The methodaccording to claim 10, wherein the isolated polynucleotide is sequencedand a synthetic nucleic acid molecule is produced from the sequence ofthe isolated polynucleotide.
 14. The method according to claim 13,wherein the synthetic nucleic acid molecule comprises the human VH, thehuman CH1, a human CH2 and a human CH3.
 15. The method of claim 1,further comprising isolating a polynucleotide encoding a variable regionof the chimeric Ig heavy chain from B cells of the transgenic mouse. 16.The method of claim 15, further comprising recombinantly making avariable region polypeptide encoded by the isolated polynucleotide. 17.The method according to claim 16, wherein recombinantly making comprisesan antibody display system, a eukaryotic expression system, aprokaryotic expression system, or an in vitro translation system. 18.The method according to claim 15, wherein the isolated polynucleotide issequenced and a synthetic nucleic acid molecule is produced from thesequence of the isolated polynucleotide.
 19. The method according toclaim 18, wherein the synthetic nucleic acid molecule comprises thehuman VH, the human CH1, a human CH2 and a human CH3.